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。
The eeBxb1 designed for expressing evolved and engineered Bxb1 in the mammalian cells
- 载体名称:
- eeBxb1
- 载体抗性:
- Ampicillin
- 载体长度:
- 5801 bp
- 载体类型:
- Mammalian Expression Vectors
- 启动子:
- CMV
- 感受态:
- Top10
- 培养温度:
- 37℃
eeBxb1 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。
eeBxb1 载体序列
LOCUS Exported 5801 bp DNA circular SYN 06-SEP-2024
DEFINITION Plasmid for expressing evolved and engineered Bxb1 in the mammalian
cells.
ACCESSION .
VERSION .
KEYWORDS eeBxb1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5801)
AUTHORS Pandey S, Gao XD, Krasnow NA, McElroy A, Tao YA, Duby JE, Steinbeck
BJ, McCreary J, Pierce SE, Tolar J, Meissner TB, Chaikof EL, Osborn
MJ, Liu DR
TITLE Efficient site-specific integration of large genes in mammalian
cells via continuously evolved recombinases and prime editing.
JOURNAL Nat Biomed Eng. 2024 Jun 10. doi: 10.1038/s41551-024-01227-1.
PUBMED 38858586
REFERENCE 2 (bases 1 to 5801)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat Biomed
Eng. 2024 Jun 10. doi: 10.1038/s41551-024-01227-1."
FEATURES Location/Qualifiers
source 1..5801
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 1..589
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 490..509
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 743..760
/label=L4440
/note="L4440 vector, forward primer"
promoter complement(834..938)
/gene="bla"
/label=AmpR promoter
primer_bind 1006..1024
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 1051..1081
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
protein_bind 1089..1105
/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)."
primer_bind 1094..1116
/label=M13/pUC Reverse
/note="In lacZ gene"
enhancer 1135..1438
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1439..1642
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 1593..1613
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 1639..1663
/label=LNCX
/note="Human CMV promoter, forward primer"
promoter 1758..1776
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 1758..1775
/label=SP6
/note="SP6 promoter, forward primer"
CDS 1814..3316
/codon_start=1
/transl_table=11
/gene="Bxb1"
/product="Bxb1 integrase"
/label=Bxb1 integrase
/note="Bxb1 large serine integrase from Mycobacterium
bacteriophage Bxb1 (Ghosh et al. 2003)."
/protein_id="AFF61396.1"
/translation="MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLD
VSGAVDPFDRKRRPNLARWLAFEEQPFDAIVAYRVDRLTRSIRHLQQLVHWAEDHKKLV
VSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGY
LPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQG
REPQGRKWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAE
LVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTV
AMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAIELAEVNAELVDLTSLIGSPAYRAG
SPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSM
NVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
polyA_signal 3728..3862
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3765..3784)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 3819..3838
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 3988..4442
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4075..4094)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4284..4305
/label=F1ori-F
/note="F1 origin, forward primer"
CDS 4729..5589
/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"
primer_bind complement(4947..4966)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
protein_bind complement(5691..5724)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."