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:
- pC0056 - LwCas13a-msfGFP-NES
- 载体抗性:
- Ampicillin
- 载体长度:
- 13585 bp
- 载体类型:
- Mammalian Expression, Lentiviral
- 复制子:
- ori
- 拷贝数:
- High Copy
- 启动子:
- EF-1α
pC0056 - LwCas13a-msfGFP-NES 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。
pC0056 - LwCas13a-msfGFP-NES 载体序列
LOCUS V000435 13585 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V000435
VERSION V000435
KEYWORDS pC0056 - LwCas13a-msfGFP-NES
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 13585)
AUTHORS Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J,
Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES,
Voytas DF, Ting AY, Zhang F
TITLE RNA targeting with CRISPR-Cas13.
JOURNAL Nature. 2017 Oct 4. doi: 10.1038/nature24049.
PUBMED 28976959
REFERENCE 2 (bases 1 to 13585)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 13585)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature.
2017 Oct 4. doi: 10.1038/nature24049."
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..13585
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(47..66)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 238..617
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 618..820
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2098..2142
/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 2291..2332
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2440..2557
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2682..3860
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 3915..7370
/label="LwCas13a"
/note="Leptotrichia wadei CRISPR-Cas effector that acts as
an RNA-guided RNAse (Abudayyeh et al., 2017)"
CDS 7416..8129
/label="superfolder GFP"
/note="GFP variant that folds robustly even when fused to
poorly folded proteins (Pedelacq et al., 2006)"
CDS 8169..8195
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
CDS 8196..8222
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
CDS 8223..8249
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
misc_feature 8277..8865
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8868..8884)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8869..8885)
/label="pBluescriptKS"
/note="For pBluescript vector"
LTR 9390..9570
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 9602..9826
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 9872..10300
/direction=RIGHT
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9959..9978)
/label="F1ori-R"
/note="F1 origin, reverse primer"
primer_bind 10169..10190
/label="F1ori-F"
/note="F1 origin, forward primer"
promoter 10314..10643
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 10691..10738
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 10757..11128
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 11261..11394
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(11431..11447)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11431..11447)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11444..11466)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 11455..11471
/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(11479..11509)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(11524..11545)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11662..11679)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(11833..12421)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12595..13452)
/label="AmpR"
/note="beta-lactamase"
promoter complement(13453..13557)
/label="AmpR promoter"