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載體描述

pcDNA3.1(+) and pcDNA3.1(-) are 5.4 kb vectors derived from pcDNA3 and designed for high-level stable and transient expression in mammalian hosts. High-level stable and non-replicative transient expression can be carried out in most mammalian cells. The vectors contain the following elements:

l Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells

l Multiple cloning sites in the forward (+) and reverse (-) orientations to facilitate cloning

l Neomycin resistance gene for selection of stable cell lines

l Episomal replication in cells lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g. COS-1, COS-7). The control plasmid, pcDNA3.1/CAT, is included for use as a positive control for transfection and expression in the cell line of choice.

l This pcDNA?3.1(+)vector is designed for high-level, constitutive expression in a variety of mammalian cell lines. It contains a Geneticin? selectable marker and a forward-orientation multiple cloning site.

The pcDNA?3.1 Expression Vector Family

Three untagged versions of pcDNA?3.1 (available separately), each with a different selectable marker (Geneticin?, Zeocin?, or Hygromycin), are for use alone or in co-transfections. All three vectors offer the following features:

l Cytomegalovirus (CMV) enhancer-promoter for high-level expression

l Large multiple cloning site in either forward (+) or reverse (-) orientations

l Bovine Growth Hormone (BGH) polyadenylation signal and transcription termination sequence for enhanced mRNA stability

l SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen (i.e., COS-1 and COS-7)

l Ampicillin resistance gene and pUC origin for selection and maintenance in E. coli.

載體序列

LOCUS       Exported                5428 bp ds-DNA     circular SYN 19-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    pCDNA3.1(+)

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 5428)

  AUTHORS   BT-lab

  TITLE     Direct Submission

  JOURNAL   Exported Tuesday, Mar 19, 2019 from SnapGene Viewer 4.3.4

            http://bt-lab.cn/

FEATURES             Location/Qualifiers

     source          1..5428

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     enhancer        235..614

                     /label=CMV enhancer

                     /note="human cytomegalovirus immediate early enhancer"

     promoter        615..818

                     /label=CMV promoter

                     /note="human cytomegalovirus (CMV) immediate early

                     promoter"

     primer_bind     829..848

                     /label=pcDNA3.1-F

     promoter        863..881

                     /label=T7 promoter

                     /note="promoter for bacteriophage T7 RNA polymerase"

     misc_feature    895..1002

                     /label=MCS

                     /note="Multiple cloning site"

     primer_bind     complement(1022..1039)

                     /label=pcDNA3.1-R

     polyA_signal    1028..1252

                     /label=bGH poly(A) signal

                     /note="bovine growth hormone polyadenylation signal"

     rep_origin      1298..1726

                     /direction=RIGHT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow

                     indicates direction of (+) strand synthesis"

     promoter        1740..2069

                     /label=SV40 promoter

                     /note="SV40 enhancer and early promoter"

     rep_origin      1920..2055

                     /label=SV40 ori

                     /note="SV40 origin of replication"

     CDS             2136..2930

                     /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

                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA

                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"

     polyA_signal    3104..3225

                     /label=SV40 poly(A) signal

                     /note="SV40 polyadenylation signal"

     primer_bind     complement(3274..3290)

                     /label=M13 rev

                     /note="common sequencing primer, one of multiple similar

                     variants"

     protein_bind    3298..3314

                     /label=lac operator

                     /bound_moiety="lac repressor encoded by lacI"

                     /note="The lac repressor binds to the lac operator to

                     inhibit transcription in E. coli. This inhibition can be

                     relieved by adding lactose or

                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."

     promoter        complement(3322..3352)

                     /label=lac promoter

                     /note="promoter for the E. coli lac operon"

     protein_bind    3367..3388

                     /label=CAP binding site

                     /bound_moiety="E. coli catabolite activator protein"

                     /note="CAP binding activates transcription in the presence

                     of cAMP."

     rep_origin      complement(3676..4261)

                     /direction=LEFT

                     /label=ori

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

                     replication"

     CDS             complement(4432..5292)

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /label=AmpR

                     /note="confers resistance to ampicillin, carbenicillin, and

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     promoter        complement(5293..5397)

                     /gene="bla"

                     /label=AmpR promoter

ORIGIN

        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg

       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg

      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc

      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt

      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata

      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc

      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc

      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt

      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt

      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca

      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg

      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc

      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg

      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca

      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc

      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc

      961 agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca

     1021 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc

     1081 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg

     1141 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg

     1201 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag

     1261 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta

     1321 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg

     1381 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa

     1441 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc

     1501 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt

     1561 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca

     1621 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc

     1681 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg

     1741 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca

     1801 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa

     1861 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca

     1921 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt

     1981 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag

     2041 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg

     2101 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg

     2161 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa

     2221 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg

     2281 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt

     2341 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa

     2401 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc

     2461 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg

     2521 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg

     2581 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg

     2641 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg

     2701 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact

     2761 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg

     2821 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc

     2881 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct

     2941 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac

     3001 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat

     3061 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc

     3121 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc

     3181 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc

     3241 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg

     3301 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg

     3361 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc

     3421 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt

     3481 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct

     3541 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga

     3601 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc

     3661 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg

     3721 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg

     3781 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt

     3841 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt

     3901 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg

     3961 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact

     4021 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt

     4081 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct

     4141 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac

     4201 cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca

     4261 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta

     4321 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa

     4381 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg

     4441 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg

     4501 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc

     4561 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc

     4621 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa

     4681 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc

     4741 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg

     4801 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc

     4861 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat

     4921 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg

     4981 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc

     5041 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg

     5101 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat

     5161 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg

     5221 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg

     5281 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct

     5341 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac

     5401 atttccccga aaagtgccac ctgacgtc

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