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查看更多产品信息 PUTATIVE FULL-LENGTH CLONE - FAQs (FL1001)
10 个常见问题解答
抱歉，我们不提供pCMVSPORT6空载体。
我们提供的cDNA克隆包含Ultimate ORF克隆，全长人类cDNA克隆，MGC克隆及GeneStorm 直接表达克隆。Ultimate ORF克隆，全长人类cDNA克隆和GeneStorm直接表达克隆是我们内部生产的，而MGC克隆是从外部获得并由ThermoFisher Scientific分销的。Ultimate ORF克隆全部经过测序并保证在氨基酸水平100%匹配GenBank序列信息，而我们提供的其它cDNA克隆没有此担保。
用于人类全长cDNA克隆库的骨架载体为pCMV-SPORT6。它是基于Gateway克隆技术的哺乳动物表达载体，插入片段由CMV启动子驱动并在其两侧插入了attB1和attB2位点。构建的重组子可直接在哺乳动物细胞中进行基因的表达。
如果您主要考虑的是质量和序列，我们推荐选择Ultimate ORF克隆。此外，这些克隆采用Gateway载体骨架，插入片段能方便地在多种宿主系统间穿梭。全长人类cDNA克隆和MGC克隆仅仅是5’和3’末端测过序的，但是全长人类cDNA克隆的优点是目的基因克隆在Gateway入门载体的骨架中。MGC克隆可提供人、小鼠和大鼠的；Ultimate ORF克隆可提供人和小鼠的，全长人类cDNA克隆只有人的。
Sorry, we do not offer the empty pCMVSPORT6 vector.
The cDNA clones we offer are Ultimate ORF clones, Full-Length human cDNA clones, MGC clones, and GeneStorm Expression-Tested clones. The Ultimate ORF clones, Full-length human cDNA clones, and the GeneStorm Expression-Ready clones are made in-house, whereas the MGC clones are derived from external sources and distributed by Thermo Fisher Scientific. The Ultimate ORF clones are fully-sequenced and guaranteed to match GenBank sequence information 100% at the amino acid level, whereas the sequence of the other cDNA clones we offer is not guaranteed.
The backbone vector that is used for the Full-length human cDNA clone collection is pCMV-SPORT6. It is a Gateway-adapted mammalian expression vector where the insert is driven by the CMV promoter and flanked by attB1 and attB2 sites. The construct can be directly used for expression of the gene in mammalian cells.
We recommend choosing the Ultimate ORF clones if quality and sequence are your main priority. Further, these clones are provided in a Gateway vector backbone, so it is easy to shuttle the insert into multiple host systems. The Full-length human cDNA clones and MGC clones are only 5´ and 3´ end-sequenced, but Full-length human cDNA clones offer the advantage of being in a Gateway Entry vector backbone. MGC clones are available for human, mouse, and rat species; Ultimate ORF clones are available for human and mouse species and Full-Length human cDNA clones are available only for human species.
These clones are not fully Gateway compatible. While the inserts are flanked by attB sites and can be transferred via BP reaction into Donor vectors, they are less than suitable for subsequent transfer into destination (DEST) vectors for protein expression, for the following reasons:
1. The inserts generally include a stop codon; hence C-terminal tagged DEST vectors are useless unless the intent is to express the insert in its native form (untagged).
2. The clones contain 5' and 3' untranslated regions of undetermined length, i.e. inserts were not specifically cloned into the Gateway reading frame. Even if the gene is in frame with a tag, the untranslated region would result in potentially several extra amino acids between the tag and the insert.
3. The 5' untranslated regions of the inserts may not contain a ribosome binding sequence (RBS) located 5-13 bases upstream of the ATG (a requirement for bacterial expression). While this does not apply to N-terminal tagged DEST vectors, which contain an RBS and ATG, it does apply to the use of C-terminal tagged vectors or vectors for native protein expression since they have no provision for an RBS upstream of the ATG.
In short, cDNA clones generated in pCMVSport6 can be transferred into mammalian DEST vectors for untagged native expression, or N-terminal tagged expression if the insert is found to already be in the Gateway reading frame (caveat - untranslated region will code for several amino acids). But for most other purposes, inserts should be amplified by PCR with primers directed at the ATG and stop codon regions and cloned into the appropriate pENTR/D-TOPO vector, or restriction-cloned in frame into the appropriate supercoiled pENTR vector.
In addition to the CAAT and TATA boxes, the CMV promoter in pcDNA3.1 vector contains sequence homologous to base pairs 137 to 724 of the sequence submitted by Boshart, et al (GenBank Accession # K03104). The complete enhancer region is contained between 214 and 620 of this sequence. Therefore, by this definition, the CMV promoter could be said to be "complete".
Please note that this promoter does not contain an intron. Some people believe that the complete promoter must contain the intron, but that has not been demonstrated to be necessary for expression.