NovoBuilder 质粒设计

pAAV-RC 质粒 (编号: V009666)

pAAV-RC7327 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300660069007200SK primerFRTProtein Rep78Capsid protein VP1M13 fwdf1 oriAmpR promoterAmpRoriCAP binding sitelac promoterlac operatorM13 rev
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基本信息

备注：pAAV-RC is an AAV helper plasmid (Agilent) encoding AAV2 Rep and Cap proteins, used in the three-plasmid AAV Helper-Free System for recombinant AAV2 production in HEK293 cells.
载体名称
pAAV-RC
载体抗性
Ampicillin
载体长度
7327 bp
载体类型
Cloning vector
复制子
ori
载体来源
Grafsky AJ III.
启动子
lac
感受态
DH10B

质粒操作方法

质粒复溶方法

  1. 发货形式：质粒干粉，常温运输，收到后请于 -20 ℃ 保存，先转化提质粒再使用。
  2. 收到干粉后先 5000 rpm 离心 1 min，加入 20 μl ddH₂O 溶解。质粒复测浓度与标称值可能有差距，这通常来自干粉在管中的位置、复溶效率、测量偏差与管壁吸附，建议先转化提质粒后再使用。
  3. 取 1 支 100 μl 感受态细胞冰上解冻 10 min，加入 2 μl 质粒，冰浴 30 min，42 ℃ 热激 60 s（不要摇动），再冰浴 2 min。
  4. 加入 900 μl 无抗生素 LB 液体培养基，180 rpm、37 ℃ 振荡培养 45 min（30 ℃ 培养 1–1.5 h）。
  5. 6000 rpm 离心 5 min，保留约 100 μl 上清重悬菌体，涂布到对应抗性的 LB 平板。
  6. 平板倒置 37 ℃ 培养 14 h（30 ℃ 则 20 h）。菌落过多可稀释质粒后再转化；无菌落可增加至 10 μl 质粒。建议先转克隆感受态、重提质粒后再导入表达感受态。
  7. 挑取单菌落接种 LB 液体培养基，加入对应抗生素，220 rpm 振荡培养 14 h，按试剂盒说明提取质粒。

通用质粒转化方法

  1. 取100 μL感受态细胞置于冰上解冻10 min，加入2 μL质粒，冰浴30 min；随后42 ℃热激60 s，期间不要晃动，再冰浴2 min。
  2. 加入900 μL无抗LB液体培养基，37 ℃、180 rpm振荡培养45 min（30 ℃条件下则培养1–1.5 h）。
  3. 6000 rpm离心5 min，弃上清，仅保留100 μL上清重悬菌体沉淀，涂布于对应质粒抗性的LB平板上。
  4. 平板倒置培养，37 ℃培养14 h，或30 ℃培养20 h。
  5. 挑取单菌落至LB液体培养基中，加入对应抗生素，220 rpm振荡培养14 h；根据实验需求及质粒提取试剂盒说明书提取质粒。

参考文献

  • Xiao X, Li J, Samulski RJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol. 1998 Mar; 72(3):2224-32. doi: 10.1128/JVI.72.3.2224-2232.1998. PMID: 9499080.
  • Collaco RF, Cao X, Trempe JP. A helper virus-free packaging system for recombinant adeno-associated virus vectors. Gene. 1999 Oct 01; 238(2):397-405. doi: 10.1016/s0378-1119(99)00347-9. PMID: 10570967.
  • Chirico J, Trempe JP. Optimization of packaging of adeno-associated virus gene therapy vectors using plasmid transfections. J Virol Methods. 1998 Dec; 76(1-2):31-41. doi: 10.1016/s0166-0934(98)00120-7. PMID: 9923737.

pAAV-RC 质粒 (编号: V009666) 注释

LOCUS       V009666                 7327 bp    DNA     circular SYN 17-DEC-2018
DEFINITION  Exported.
ACCESSION   V009666
VERSION     V009666
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 7327)
  AUTHORS   Grafsky AJ III.
  TITLE     pAAV-RC for AAV Helper-Free System
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7327)
  AUTHORS   Grafsky AJ III.
  TITLE     Direct Submission
  JOURNAL   Submitted (16-APR-2001) Technical Services, Stratagene, 11011 N.
            Torrey Pines Rd., La Jolla, CA 92037, USA
REFERENCE   3  (bases 1 to 7327)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7327)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName:
            "Unpublished"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (16-APR-2001) Technical Services, Stratagene, 11011 N. Torrey Pines
            Rd., La Jolla, CA 92037, USA"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..52
                     /label="SK primer"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    66..113
                     /label="FRT"
                     /note="FLP-mediated recombination occurs in the 8-bp core
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             131..1993
                     /gene="Rep78"
                     /label="Protein Rep78"
                     /note="Protein Rep78 from Adeno-associated virus 2 (isolate
                     Srivastava/1982). Accession#: Q89268"
     CDS             2013..4217
                     /gene="VP1"
                     /label="Capsid protein VP1"
                     /note="Capsid protein VP1 from Adeno-associated virus 2
                     (isolate Srivastava/1982). Accession#: P03135"
     primer_bind     complement(4548..4564)
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     rep_origin      complement(4705..5160)
                     /direction=LEFT
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5187..5291
                     /label="AmpR promoter"
     CDS             5292..6149
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      6323..6911
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     protein_bind    7199..7220
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        7235..7265
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    7273..7289
                     /label="lac operator"
                     /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     7297..7313
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"