我们所展示的质粒图谱主要是从文献和开放数据库中收集而来，主要是为了方便研究工作，其中一小部分质粒进行了质量控制，可供科学家使用。
所有的产品都严格仅供科学研究使用，不能应用于临床试验，包括人体摄入、注射或外用的药理学用途。
确保质粒的关键元件正确，但是我们并不能保证实验结果。页面展示的图谱序列为理论序列，可能与测序结果不一致，请自行比对后确定是否满足要求。(如果测过序，本页面一般会提供下载)
开放数据库中的大多数载体序列都没有被完全测序。如果实际序列与参考序列的相似度超过99%，则将其视为正确。
由于科学研究是在探索未知，具有很大的不确定性，在任何情况下，我们都不承担超出质粒本身的额外经济损失或责任。
- 载体名称:
- pInducer10-mir-RUP-PheS
- 载体抗性:
- Ampicillin
- 载体长度:
- 14567 bp
- 载体类型:
- Mammalian Expression, Lentiviral
- 复制子:
- ori
- 筛选标记:
- Puromycin
- 启动子:
- UbC
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- LNCX_primer
pInducer10-mir-RUP-PheS 载体图谱
质粒操作方法
1. 发货形式：质粒干粉(常温运输，存于-20度，请务必先转化提质粒后使用)
2. 收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O溶解质粒;（质粒复测的浓度有时候与标称值差距较大，这可能是因为冻干质粒在管中的位置、复溶效率、测量偏差以及管壁的吸附导致，因此建议先转化提质粒后再使用）
3. 取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；
4. 加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min (30℃培养1-1.5小时);
5. 6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上;
6. 将平板倒置37℃培养14h，如果要求是30℃则培养20h; (菌落过多则将质粒稀释后再转化；没有菌落则加入10μl质粒转化；建议不要直接转表达感受态， 要先转克隆感受态，重提质粒后再导入表达感受态);
7. 挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
pInducer10-mir-RUP-PheS 载体序列
LOCUS V010531 14567 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V010531
VERSION V010531
KEYWORDS pInducer10-mir-RUP-PheS
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14567)
AUTHORS Meerbrey KL, Hu G, Kessler JD, Roarty K, Li MZ, Fang JE,
Herschkowitz JI, Burrows AE, Ciccia A, Sun T, Schmitt EM, Bernardi
RJ, Fu X, Bland CS, Cooper TA, Schiff R, Rosen JM, Westbrook TF,
Elledge SJ
TITLE The pINDUCER lentiviral toolkit for inducible RNA interference in
vitro and in vivo.
JOURNAL Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3665-70. doi:
10.1073/pnas.1019736108. Epub 2011 Feb 9.
PUBMED 21307310
REFERENCE 2 (bases 1 to 14567)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14567)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1073/pnas.1019736108"; journalName: "Proc Natl Acad Sci U S A";
date: "2011-03-1- 1"; volume: "108"; issue: "9"; pages: "3665-70"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14567
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 497..730
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 915..959
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 1108..1149
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 1257..1374
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
CDS complement(1442..1813)
/label="BleoR"
/note="antibiotic-binding protein"
promoter complement(1833..1880)
/label="EM7 promoter"
/note="synthetic bacterial promoter"
protein_bind 1950..2220
/label="tetracycline response element"
/note="contains seven copies of the tetracycline operator
tetO"
promoter 2253..2291
/label="minimal CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 2288..2312
/label="LNCX"
/note="Human CMV promoter, forward primer"
CDS 2388..3080
/label="TurboRFP"
/note="red fluorescent protein from Entacmaea quadricolor"
ncRNA 3134..3227
/label="5' miR-30a"
/note="sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)"
primer_bind 3227..3243
/label="pBluescriptKS"
/note="For pBluescript vector"
primer_bind 3228..3244
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
CDS 3265..3282
/label="thrombin site"
/note="thrombin recognition and cleavage site"
CDS 3283..3306
/label="HRV 3C site"
/note="recognition and cleavage site for human rhinovirus
3C and PreScission proteases"
regulatory 3316..3325
/label="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS complement(3483..4463)
/gene="pheS"
/label="Phenylalanine--tRNA ligase alpha subunit"
/note="Phenylalanine--tRNA ligase alpha subunit from
Escherichia coli O139:H28 (strain E24377A / ETEC).
Accession#: A7ZMI2"
CDS 4601..4621
/label="TEV site"
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
ncRNA 4645..4757
/label="3' miR-30a"
/note="sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)"
promoter 4808..6019
/label="UbC promoter"
/note="human ubiquitin C promoter"
protein_bind 6026..6059
/label="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
CDS 6066..6809
/label="rtTA-Advanced"
/note="improved tetracycline-controlled transactivator"
protein_bind complement(6813..6846)
/label="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
misc_feature 6862..7444
/label="IRES2"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 7443..8036
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 8061..8649
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 8856..9089
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 9196..9420
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 9466..9894
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9903..9923)
/label="pBABE 3'"
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 10019..10229
/label="SV40 promoter"
/note="SV40 early promoter"
polyA_signal 11060..11181
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(11230..11246)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11230..11246)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11243..11265)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 11254..11270
/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(11278..11308)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(11323..11344)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11461..11478)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(11632..12220)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12394..13251)
/label="AmpR"
/note="beta-lactamase"
promoter complement(13252..13356)
/label="AmpR promoter"
polyA_signal 13404..13538
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
LTR 13706..14339
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"