质粒图谱信息

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pUC18+和+pUC19质粒图谱

pUC18 和pUC19质粒图谱pUC18 和pUC19 大小只有2686bp ，是最常用的质粒载体，其结构组成紧凑，几乎不含多余的DNA 片段，GenBank注册号为L08752（pUC18）和X02514（pUC19）。

由pBR322 改造而来，其中lacZ（MSC）来自M13mp18/19 图3-4 是其质粒图谱。

这两个质粒的结构几乎是完全一样的，只是多克隆位点的排列方向相反。

这些质粒缺乏控制拷贝数的rop基因，因此其拷贝数达500-700 。

pUC 系列载体含有一段lacZ蛋白氨基末端的部分编码序列，在特定的受体细胞中可表现α-互补作用。

因此在多克隆位点中插入了外源片段后，可通过α-互补作用形成的蓝色和白色菌落筛选重组质粒。

特征序列区位：pBR322_origin1471 - 852Ampicillin2486 - 1626lac_promoter543 - 514AmpR_promoter2556 - 2528M13_pUC_rev_primer500 - 478M13_reverse_primer479 - 461M13_forward20_primer379 - 395M13_pUC_fwd_primer364 - 386lacZ_a398 - 250pGEX_3_primer51 - 29Orf22486 - 1626NOS terminator sequence:ggatccatcgttcaaac atttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataattt ctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttgtttatgagatgggtttttatgattagagtccc gcaattatacatttaatacacgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcat ctatgt tactagtaatcgat设计引物：Forward primer: 5’ ggaGGATCC ggatccatcgttcaaac 3’(含Bam H I 位点GGATCC及三个保护碱基)Reverse primer: 5’ atcGAATTC ATCGATTACTAGTAACATAG 3’(含Eco R I 位点GAATTC及三个保护碱基)请查一下NOS终止子序列内有没有这两个酶切点。

pUC18 和 pUC19质粒图谱

wowenwenpUC18 和pUC19质粒图谱pUC18 和pUC19 大小只有2686bp ，是最常用的质粒载体，其结构组成紧凑，几乎不含多余的DNA 片段，GenBank注册号为L08752（pUC18）和X02514（pUC19）。

由pBR322 改造而来，其中lacZ（MSC）来自M13mp18/19 图3-4 是其质粒图谱。

这两个质粒的结构几乎是完全一样的，只是多克隆位点的排列方向相反。

这些质粒缺乏控制拷贝数的rop基因，因此其拷贝数达500-700 。

pUC 系列载体含有一段lacZ蛋白氨基末端的部分编码序列，在特定的受体细胞中可表现α-互补作用。

因此在多克隆位点中插入了外源片段后，可通过α-互补作用形成的蓝色和白色菌落筛选重组质粒。

质粒图谱查询方法

By
0099--pGE-1—Stratagene--RNAi载体 0100--pSUPER.p53—OligoEngine--RNAi载体 0101--palter-ex1--promega 0102--pACYCDuet-1--NOVAGEN 0103--pEX lox(+) Vector—NOVAGEN--原核表达 0104--质粒名称：pBACgus-8 Transfer Plasmid—NOVAGEN--CHUANSUO 0105--pSCREEN?-1b(+) Vector Map—novagen--筛选 0106--PGEX-2T--BD Co--pDsRed2--Clontech 0107--pbgal-Basic—Clontech--mammalian reporter vector 0108—pBI—Clontech--express two genes of interest from a bidirectional tet-responsive promoter 0109--质粒名称：pbgal-Control—Clontech--mammalian reporter vector 0110-- pGEX-5X-1--原核表达 0111--pBI-EGFP—Clontech--pBI-EGFP-- coexpress 0112--pBI-G—Clontech--pBI-G--express b-galactosidase 0113--pBI-GL—Clontech--pBI-GL --express luciferase and b-galactosidase 0114--pCMS-EGFP—Clontech--mammalian expression vector 0115--pd2EYFP-1—Clontech--启动子测定 0116--质粒名称--pd2EYFP-N1—Clontech--融合表达 0117--pd4EGFP-Bid—Clontech--融合表达 Bid 0118--pDNR-CMV—Clontech--pDNR-CMV 0119--pDNR-EGFP Vector—Clontech 0120--pDNR-LacZ –Clontech 0121--pECFP-Endo—Clontech--真核表达0122--pECFP-ER—Clontech--真核表达0123--pEGFP-Actin—Clontech--真核表达0124--pGAD GH--Clontech--酵母表达 0125--pGADT7-Rec –Clontech--酵母表达 0126--pGADT7-RecAB—Clontech--酵母表达 0127--pGADT7-Rec2—Clontech--酵母表达 0128--pGBKT7—Clontech--酵母表达 0129--pHAT 10/11/12—Clontech 0130--pHAT20—Clontech 0131—pHygEGFP—Clontech 0132—pLacZi—Clontech 0133—pM—Clontech--pM is used to generate a fusion of the GAL4 DNA-BD 0134--pPKCa-EGFP—Clontech 0135--pPKCb-EGFP—Clontec 0136--pSIREN-DNR Vector—Clontech--RNAi 0137--pSIREN-DNR-DsRed-Express Vector—Clontech--RNAi 0138--pSIREN-RetroQ—Clontech--RNAi 0139--pIRES-EYFP—Clontech--RNAi 0140--pSRE-Luc—Clontech--RNAi 0141--pTK-neo—novagen--原核表达 0142--pZsGreen Vector—Clontech--pZsGreen is a pUC19-derived prokaryotic expression vector 0143--pTandem-1—novagen--原核表达 0144--pZsGreen1-C1Vector—Clontech----真核表达 0145--质粒名称：M13mp18—novagen--原核表达 0146--pZsGreen1-DR Vector—Clontech--真核表达 0147--PZsGreen1-N1 Vector—Clontech --真核表达 0148--T7Select415-1b—novagen----真核表达 0149--pZsYellow Vector—Clontech --真核表达 0150—pTimer—Clontech --真核表达 0151--pTA-Luc—Clontech --真核表达 0152--pTAL-Luc—Clontech --真核表达 0153--pTA-SEAP—Clontech --真核表达 0154--pTAL-SEAP—Clontech --真核表达 0155--pTet-On—Clontech --真核表达 0156--pTet-Off—Clontech --真核表达 0157--pTet-ATF—Clontech --真核表达 0158--pTet-CREB—Clontech --真核表达

质粒图谱查询方法

3.google scholar: / 有些质粒是经过改造的，所以通过上述方法不能查询到相应信息。这时，可以在google scholar中输入质粒名称，可以直观地看哪些学者在何文章中使用了该质粒，从而可了解到质粒的来源;或者籍此向作者咨询或索取。 4.尝试从各大生物公司，例如invitrogen网站查询. 5. 这个网站收录了大量图谱： http://www.embl-hamburg.de/~geerlof/webPP/vectordb/bact_vectors/table.html
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file:///D|/中科院/Selective Serotonin Transporter/质粒信息/质粒图谱查询方法.txt
file:///D|/中科院/Selective Serotonin Transporter/质粒信息/质粒图谱查询方法.txt（第 2／6 页）[2011/8/4 18:39:52]
By
0099--pGE-1—Stratagene--RNAi载体 0100--pSUPER.p53—OligoEngine--RNAi载体 0101--palter-ex1--promega 0102--pACYCDuet-1--NOVAGEN 0103--pEX lox(+) Vector—NOVAGEN--原核表达 0104--质粒名称：pBACgus-8 Transfer Plasmid—NOVAGEN--CHUANSUO 0105--pSCREEN?-1b(+) Vector Map—novagen--筛选 0106--PGEX-2T--BD Co--pDsRed2--Clontech 0107--pbgal-Basic—Clontech--mammalian reporter vector 0108—pBI—Clontech--express two genes of interest from a bidirectional tet-responsive promoter 0109--质粒名称：pbgal-Control—Clontech--mammalian reporter vector 0110-- pGEX-5X-1--原核表达 0111--pBI-EGFP—Clontech--pBI-EGFP-- coexpress 0112--pBI-G—Clontech--pBI-G--express b-galactosidase 0113--pBI-GL—Clontech--pBI-GL --express luciferase and b-galactosidase 0114--pCMS-EGFP—Clontech--mammalian expression vector 0115--pd2EYFP-1—Clontech--启动子测定 0116--质粒名称--pd2EYFP-N1—Clontech--融合表达 0117--pd4EGFP-Bid—Clontech--融合表达 Bid 0118--pDNR-CMV—Clontech--pDNR-CMV 0119--pDNR-EGFP Vector—Clontech 0120--pDNR-LacZ –Clontech 0121--pECFP-Endo—Clontech--真核表达0122--pECFP-ER—Clontech--真核表达0123--pEGFP-Actin—Clontech--真核表达0124--pGAD GH--Clontech--酵母表达 0125--pGADT7-Rec –Clontech--酵母表达 0126--pGADT7-RecAB—Clontech--酵母表达 0127--pGADT7-Rec2—Clontech--酵母表达 0128--pGBKT7—Clontech--酵母表达 0129--pHAT 10/11/12—Clontech 0130--pHAT20—Clontech 0131—pHygEGFP—Clontech 0132—pLacZi—Clontech 0133—pM—Clontech--pM is used to generate a fusion of the GAL4 DNA-BD 0134--pPKCa-EGFP—Clontech 0135--pPKCb-EGFP—Clontec 0136--pSIREN-DNR Vector—Clontech--RNAi 0137--pSIREN-DNR-DsRed-Express Vector—Clontech--RNAi 0138--pSIREN-RetroQ—Clontech--RNAi 0139--pIRES-EYFP—Clontech--RNAi 0140--pSRE-Luc—Clontech--RNAi 0141--pTK-neo—novagen--原核表达 0142--pZsGreen Vector—Clontech--pZsGreen is a pUC19-derived prokaryotic expression vector 0143--pTandem-1—novagen--原核表达 0144--pZsGreen1-C1Vector—Clontech----真核表达 0145--质粒名称：M13mp18—novagen--原核表达 0146--pZsGreen1-DR Vector—Clontech--真核表达 0147--PZsGreen1-N1 Vector—Clontech --真核表达 0148--T7Select415-1b—novagen----真核表达 0149--pZsYellow Vector—Clontech --真核表达 0150—pTimer—Clontech --真核表达 0151--pTA-Luc—Clontech --真核表达 0152--pTAL-Luc—Clontech --真核表达 0153--pTA-SEAP—Clontech --真核表达 0154--pTAL-SEAP—Clontech --真核表达 0155--pTet-On—Clontech --真核表达 0156--pTet-Off—Clontech --真核表达 0157--pTet-ATF—Clontech --真核表达 0158--pTet-CREB—Clontech --真核表达

pPICZαA质粒图谱

pPICZaa载体基本信息出品公司: InvitrogenpPICZalpha A, pPICZαA, pPICZalphaA, pPICZαA, pPICZaA, 载体名称:pPICZa-A质粒类型: 毕赤酵母蛋白表达载体表达水平: 高拷贝启动子: AOX1克隆方法: 多克隆位点，限制性内切酶载体大小: 3593 bp5' 测序引物及序列: 5´AOX1:5´-GACTGGTTCCAATTGACAAGC-3´3' 测序引物及序列: 3´AOX1:5´-GCAAATGGCATTCTGACATCC-3´载体标签: C-Myc, C-His载体抗性: Zeocin 博来霉素筛选标记: HIS4备注: 插入基因是必需包含起始密码子ATG。

产品目录号: V195-20稳定性: 稳定Stable组成型: 组成型Constitutive病毒/非病毒: 非病毒pPICZaa载体质粒图谱和多克隆位点信息pPICZaa载体简介pPICZαA，B和C是3.6 kb的毕赤酵母蛋白分泌表达载体。

表达的重组蛋白是融合蛋白，含有一个N-端多肽，编码酿酒酵母（Saccharomyces cerevisiae）α-因子分泌信号。

载体能够在毕赤酵母中利用甲醇诱导的高水平的表达目的蛋白，并且可以用在任何毕赤酵母中，包括X33，GS115菌株，SMD1168H，KM71H。

pPICZαA，B和C系列载体包含以下元素：•5'端含有AOX1启动子的严格调控，利用甲醇诱导表达任何感兴趣的基因（Ellis等，1985; Koutz 等人，1989;tschopp等人，1987A）。

•α-因子分泌信号能够分泌性表达目的蛋白。

•Zeocin抗性基因在大肠杆菌(Escherichia coli)和毕赤酵母都能用于筛选（Baron等人,1992; Drocourt等人，1990）。

质粒图谱

)质粒图谱登记号：00012)质粒名称：pIRES3)来源：BD Co4)用途：真核双表达5)是否可以提供更详细资料：可以6)是否可以共享：7)联系方式：PM)质粒图谱登记号：00022)质粒名称：pECFP-C13)来源：BD Co4)用途：检测真核表达5)是否可以提供更详细资料：可以6)是否可以共享：7)联系方式：pm1)质粒图谱登记号：00032)质粒名称：pShuttle3)来源：4)用途：5)是否可以提供更详细资料：6)是否可以共享：7)联系方式：2)pShuttle MCS很多人用pEGFP-C1，我也来发一个)质粒图谱登记号：00042)质粒名称：pSBR322、pUC183)来源：4)用途：5)是否可以提供更详细资料：6)是否可以共享：7)联系方式：1)质粒图谱登记号：00052)质粒名称：pcDNA3.1(+)/CAT3)来源：invitrogen4)用途：真核表达5)是否可以提供更详细资料：可以6)是否可以共享：无偿7)联系方式：PM1)质粒图谱登记号：00062)质粒名称：pQEx3)来源：Qiagen4)用途：原核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM2)1)质粒图谱登记号：00072)质粒名称：pIVEX2.33)来源：Rocho4)用途：体外转录翻译5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM质粒图谱登记号：0008质粒名称：pIRES-EGFP来源：用途：是否可以提供更详细资料：是否可以共享：否联系方式：1)质粒图谱登记号：00092)质粒名称：pET-28a（+）3)来源：Novagen4)用途：原核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM1)质粒图谱登记号：00112)质粒名称：pET-32a(+)3)来源：novagen4)用途：原核表达5)是否可以提供更详细资料：6)是否可以共享：7)联系方式：pm1)质粒图谱登记号：00132)质粒名称：pcDNA3.1/Zeo (+)3)来源：invitrogen4)用途：原核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM)质粒图谱登记号：00132)质粒名称：pEGFP-N33)来源：clontech4)用途：真核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM)质粒图谱登记号：00142)质粒名称：pcDNA33)来源：invitrogen4)用途：真核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM)质粒图谱登记号：00152)质粒名称：pfastbac13)来源：invitrogene4)用途：昆虫表达5)是否可以提供更详细资料：不可以6)是否可以共享：不可以7)联系方式：PM)质粒图谱登记号：00162)质粒名称：pEGFP-C33)来源：clontech4)用途：真核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PMwangjun2002274 edited on 2004-06-22 00:041)质粒图谱登记号：00172)质粒名称：pSecTag23)来源：Invitrogen4)用途：真核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM)1质粒图谱登记号：00192)质粒名称：pET20b3)来源：NOVAGEN4)用途：原核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM1)质粒图谱登记号：00222)质粒名称：pThioHisA3)来源：invitrogen4)用途：原核表达5)是否可以提供更详细资料：4.365kb , HP-thioredoxin fusion proteinexpressionvector, trc promoter, Ampr, a EK cleavage site lies between HP-thioredoxin and MCS宿主菌TOP10（基因型为：F-mcrA △（mrr-hsd RMS-mcrBC）Ф80 lacZ M15 △lacX74 deoR recAl araD139 △（ara-leu）7697 galU galK rpsL endAl nupG6)是否可以共享：交换或其它7)联系方式：PM2)3)1)质粒图谱登记号：00242)质粒名称：pcDNA3.1-Myc-His-A-3)来源：invitrogen4)用途：真核核表达4))质粒图谱登记号：00252)质粒名称：pSUPER.neo3)来源：4)用途：siRNA5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM)质粒图谱登记号：00312)质粒名称：pSilencer1.0-siRNA3)来源：Ambion4)用途：RNAi5)是否可以提供更详细资料：/techlib/Documents.html?fkResSxn=7&fkSub Sxn=236)是否可以共享：实验结束后，可提供含shRNA模板的质粒7)联系方式：pm5) )质粒图谱登记号：00322)质粒名称：pSilencer2.0-U6siRNA 3)来源：Ambion4)用途：RNAi ，与1.0相比，可以建立稳转株5)更详细资料：/techlib/prot/fm_7209.pdf 6)是否可以共享：交换 7)联系方式：pm6)会员名：mlluoE-mail：*************可提供试验资源名称和简要介绍：pSilencer 3.1-H1 neo Vector，是Ambion公司目前最高版本的shRNA 载体，为扩增此载体我已插入目的片段，如有战友需要，将此片段双酶切再连上自己的片段即可。

质粒图谱大全

（转载）一. 九种表达载体Pllp-OmpA, pllp-STII, pMBP-P, pMBP-C,pET-GST, pET-Trx, pET-His, pET-CKS, pET-DsbA二. 克隆载体pTZ19RDNApUC57DNAPMD18TPQE30pUC18pUC19pTrcHisApTrxFuspRSET-ApRSET-BpVAX1PBR322pbv220pBluescriptIIKS( )L4440pCAMBIA-1301pMAL-p2XpGD926三.PET 系列表达载体ProteinExpression?ProkaryoticExpression?pETDsbFusionSystems39band40b ProteinExpression?ProkaryoticExpression?pETExpressionSystem33b ProteinExpression?ProkaryoticExpression?pETExpressionSystems ProteinExpression?ProkaryoticExpression?pETExpressionSystemsplusCompetentCells ProteinExpression?ProkaryoticExpression?pETGSTFusionSystems41and42 ProteinExpression?ProkaryoticExpression?pETNusAFusionSystems43.1and44 ProteinExpression?ProkaryoticExpression?pETVectorDNAProteinPurification?PurificationSystems?Strep?TactinResinsandPurificationKits四.PGEX 系列表达载体TEcoR?pGEX-1I/BAPpGEX-2TpGEX-2TKpGEX-3XpGEX-4T-1pGEX-4T-2pGEX-4T-3pGEX-5X-1pGEX-5X-2pGEX-5X-3pGEX-6P-1pGEX-6P-2pGEX-6P-3五.PTYBsystemPTYB1PTYB2PTYB11PTYB12六. 真核表达载体pCDNA3.1(-)pCDNA3.1( )pPICZalphaApGAPZα APYES2.0pBI121pEGFP-N1pEGFP-C1pPIC9KpPIC3.5K如何阅读分析质粒图谱载体主要有病毒和非病毒两大类, 其中质粒DNA是一种新的非病毒转基因载体。

质粒

pCMV-C-Flag质粒图谱pCMV-C-Flag质粒图谱pCMV-C-Flag的MCS：XmaI PstISacI Small BamHI HindIII651 GAGCTCCACC GCGGTGGCGG CCGCTCTAGC CCGGGCGGAT CCAAGCTTCTCTCGAGGTGG CGCCACCGCC GGCGAGATCG GGCCCGCCTA GGTTCGAAGAFlagEcoRI EcoRV SacI BglII XhoI XbaI D Y K D701 GCAGGAATTC GATATCGTCG ACAGATCTCT CGAGTCTAGA GATTACAAGGCGTCCTTAAG CTATAGCAGC TGTCTAGAGA GCTCAGATCT CTAATGTTCCtagD D D K ApaI751 ATGACGACGA TAAGTAAGGG CCCGGTACCT TAATTAATTA AGGTACCAGGTACTGCTGCT ATTCATTCCC GGGCCATGGA ATTAATTAAT TCCATGGTCC说明：1.该质粒的大小为4317bp2. Ori控制复制起始的位点，该质粒有两个复制位点，分别是：pUCori f1ori，是真核质粒。

3.该质粒图谱上的抗生素抗性基因有： Neor r/kanr4.该质粒的多克隆位点是MCS Flag tag，克隆携带外源基因片段。

5.该质粒有启动子pSV40,p bla,pCMV，可以使目的基因在增殖的细胞中稳定表达。

6.该质粒TkpA、SV40pA信号是加polyA信号，可以起到稳定mRNA的作用。

综上所述，该质粒有质粒所需的组成元素，是一个合格的质粒。

基因工程技术流程图。

pcDNA3.1质粒图谱

pcDNA3.1(+)pcDNA3.1(-)Catalog nos. V790-20 and V795-20, respectivelyVersion I08140128-0104tech_service@iiTable of ContentsTable of Contents (iii)Important Information (v)Purchaser Notification (vi)Methods (1)Overview (1)Cloning into pcDNA3.1 (2)Transfection (6)Creation of Stable Cell Lines (7)Appendix (10)pcDNA3.1 Vectors (10)pcDNA3.1/CAT (12)Technical Service (13)References (15)iiiivImportant InformationContents pcDNA3.1 is supplied as follows:Catalog no.ContentsV790-2020 µg pcDNA3.1(+), lyophilized in TE, pH 8.020 µg pcDNA3.1/CAT, lyophilized in TE, pH 8.0V795-2020 µg pcDNA3.1(-), lyophilized in TE, pH 8.020 µg pcDNA3.1/CAT, lyophilized in TE, pH 8.0 Shipping/Storage Lyophilized plasmids are shipped at room temperature and should be stored at -20°C.Product Qualification Each of the pcDNA3.1 vectors is qualified by restriction enzyme digestion with specific restriction enzymes as listed below. Restriction digests must demonstrate the correct banding pattern when electrophoresed on an agarose gel. The table below lists the restriction enzymes and the expected fragments.Vector Restriction Enzyme Expected Fragments (bp) pcDNA3.1(+)Nhe IPst ISac I54281356, 4072109, 5319pcDNA3.1(-)Nhe IPst ISac I54271363, 4064169, 5258pcDNA3.1/CAT Nhe IPst ISac I62172145, 4072109, 6008vPurchaser NotificationIntroduction Use of pcDNA3.1 is covered under a number of different licenses as described below.CMV Promoter Use of the CMV promoter is covered under U.S. Patent Nos. 5,168,062 and 5,385,839owned and licensed by the University of Iowa Research Foundation and may be used forresearch purposes only. Commercial users must obtain a license to these patents directlyfrom the University of Iowa Research Foundation. Inquiries for commercial use should bedirected to:Brenda AkinsUniversity of Iowa Research Foundation (UIRF)214 Technology Innovation CenterIowa City, IA 52242Phone:319-335-4549BGH Polyadenylation Signal The bovine growth hormone (BGH) polyadenylation sequence is licensed under U.S. Patent No. 5,122,458 for research purposes only. “Research purposes” means uses directed to the identification of useful recombinant proteins and the investigation of the recombinant expression of proteins, which uses shall in no event include any of the following:a.any use in humans of a CLAIMED DNA or CLAIMED CELL;b.any use in human of protein or other substance expressed or made at any stage of itsproduction with the use of a CLAIMED DNA or a CLAIMED CELL;c.any use in which a CLAIMED DNA or CLAIMED CELL would be sold ortransferred to another party other than Invitrogen, its AFFILIATE, or itsSUBLICENSEE;d.any use in connection with the expression or production of a product intended forsale or commercial use; ore.any use for drug screening or drug development.Inquiries for commercial use should be directed to:Bennett Cohen, Ph.D.Research Corporation Technologies101 North Wilmot Road, Suite 600Tucson, AZ 85711-3335Tel: 1-520-748-4400Fax: 1-520-748-0025viMethodsOverviewIntroduction pcDNA3.1(+) and pcDNA3.1(-) are 5.4 kb vectors derived from pcDNA3 and designed for high-level stable and transient expression in mammalian hosts. High-level stable andnon-replicative transient expression can be carried out in most mammalian cells. Thevectors contain the following elements:•Human cytomegalovirus immediate-early (CMV) promoter for high-level expressionin a wide range of mammalian cells•Multiple cloning sites in the forward (+) and reverse (-) orientations to facilitatecloning•Neomycin resistance gene for selection of stable cell lines•Episomal replication in cells lines that are latently infected with SV40 or that expressthe SV40 large T antigen (e.g. COS-1, COS-7)The control plasmid, pcDNA3.1/CAT, is included for use as a positive control fortransfection and expression in the cell line of choice.Experimental Outline Use the following outline to clone and express your gene of interest in pcDNA3.1.1.Consult the multiple cloning sites described on pages 3-4 to design a strategy to cloneyour gene into pcDNA3.1.2.Ligate your insert into the appropriate vector and transform into E. coli. Selecttransformants on LB plates containing 50 to 100 µg/ml ampicillin.3.Analyze your transformants for the presence of insert by restriction digestion.4.Select a transformant with the correct restriction pattern and use sequencing toconfirm that your gene is cloned in the proper orientation.5.Transfect your construct into the mammalian cell line of interest using your ownmethod of choice. Generate a stable cell line, if desired.6.Test for expression of your recombinant gene by western blot analysis or functionalassay.1Cloning into pcDNA3.1Introduction Diagrams are provided on pages 3-4 to help you design a cloning strategy for ligating your gene of interest into pcDNA3.1. General considerations for cloning and transformation arelisted below.General Molecular Biology Techniques For help with DNA ligations, E. coli transformations, restriction enzyme analysis, purification of single-stranded DNA, DNA sequencing, and DNA biochemistry, please refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).E. coli Strain Many E. coli strains are suitable for the propagation of this vector including TOP10F´,DH5α™-T1R, and TOP10. We recommend that you propagate vectors containing inserts inE. coli strains that are recombination deficient (rec A) and endonuclease A-deficient(end A).For your convenience, TOP10F´ is available as chemically competent or electrocompetentcells from Invitrogen.Item Quantity Catalog no.One Shot® TOP10F´ (chemically competent cells)21 x 50 µl C3030-03Electrocomp™ TOP10F´ 5 x 80 µl C665-55Ultracomp™ TOP10F´ (chemically competent cells) 5 x 300 µl C665-03Transformation Method You may use any method of your choice for transformation. Chemical transformation is the most convenient for most researchers. Electroporation is the most efficient and the method of choice for large plasmids.Maintenance of pcDNA3.1To propagate and maintain pcDNA3.1, we recommend resuspending the vector in 20 µl sterile water to make a 1 µg/µl stock solution. Store the stock solution at -20°C.Use this stock solution to transform a rec A, end A E. coli strain like TOP10F´, DH5α™-T1R, TOP10, or equivalent. Select transformants on LB plates containing 50 to100 µg/ml ampicillin. Be sure to prepare a glycerol stock of your plasmid-containing E. coli strain for long-term storage (see page 5).Cloning Considerations pcDNA3.1(+) and pcDNA3.1(-) are nonfusion vectors. Your insert must contain a Kozak translation initiation sequence and an ATG start codon for proper initiation of translation (Kozak, 1987; Kozak, 1991; Kozak, 1990). An example of a Kozak consensus sequence is provided below. Please note that other sequences are possible (see references above), but the G or A at position -3 and the G at position +4 are the most critical for function (shown in bold). The ATG initiation codon is shown underlined.(G/A)NNATG GYour insert must also contain a stop codon for proper termination of your gene. Please note that the Xba I site contains an internal stop codon (TCTAGA).continued on next page23Multiple Cloning Site of pcDNA3.1(+)Below is the multiple cloning site for pcDNA3.1(+). Restriction sites are labeled to indicate the cleavage site. The Xba I site contains an internal stop codon (TCTAGA). The multiple cloning site has been confirmed by sequencing and functional testing. Thecomplete sequence of pcDNA3.1(+) is available for downloading from our web site( ) or from Technical Service (see page 13). For a map and adescription of the features of pcDNA3.1(+), please refer to the Appendix , pages 10-11.1109TCCTTTCCTA ATAAAATGAG GAAATTGCAT CAAT TA TABGH poly (A) siteCATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCGTAACAACTCC GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATATAGTCTAGAGG GCCCGTTTAA ACCCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CATCTGTTGT TTGCCCCTCC CCCGTGCCTT CCTTGACCCT GGAAGGTGCC ACTCCCACTG6897498098699299891049enhancer region (3´ end)*Please note that there are two Bst X I sites in the polylinker.continued on next page4Multiple CloningSite ofpcDNA3.1(-)Below is the multiple cloning site for pcDNA3.1(-). Restriction sites are labeled to indicate the cleavage site. The Xba I site contains an internal stop codon (TCTAGA). The multiple cloning site has been confirmed by sequencing and functional testing. Thecomplete sequence of pcDNA3.1(-) is available for downloading from our web site ( ) or from Technical Service (see page 13). For a map and a description of the features of pcDNA3.1(-), please see the Appendix , pages 10-11.Hin d III CAAT TA TA Bam H I Bst X I*Eco R I Eco R V Bst X I*BGH poly (A) site Kpn I Afl II Pme I Asp 718 I pcDNA3.1/BGH reverse priming site CCTTTCCTAA TAAAATGAGG AAATTGCATC ATCTGTTGTT TGCCCCTCCC CCGTGCCTTC CTTGACCCTG GAAGGTGCCA CTCCCACTGT GGTACCAAGC TTAAGTTTAA ACCGCTGATC AGCCTCGACT GTGCCTTCTA GTTGCCAGCC GCCGCCACTG TGCTGGATAT CTGCAGAATT CCACCACACT GGACTAGTGG ATCCGAGCTC TAACAACTCC GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG 11091049989929869809749689enhancer region (3´ end)*Please note that there are two Bst X I sites in the polylinker.continued on next pageCloning into pcDNA3.1, continuedE. coli Transformation Transform your ligation mixtures into a competent rec A, end A E. coli strain (e.g. TOP10F´, DH5α™-T1R, TOP10) and select transformants on LB plates containing 50 to 100 µg/ml ampicillin. Select 10-20 clones and analyze for the presence and orientation of your insert.We recommend that you sequence your construct with the T7 Promoter and BGH Reverseprimers (Catalog nos. N560-02 and N575-02, respectively) to confirm that your gene is in thecorrect orientation for expression and contains an ATG and a stop codon. Please refer to thediagrams on pages 3-4 for the sequences and location of the priming sites. The primers areavailable separately from Invitrogen in 2 µg aliquots.Preparing aGlycerol StockOnce you have identified the correct clone, purify the colony and make a glycerol stock forlong-term storage. You should keep a DNA stock of your plasmid at -20°C.•Streak the original colony out on an LB plate containing 50 µg/ml ampicillin. Incubatethe plate at 37°C overnight.•Isolate a single colony and inoculate into 1-2 ml of LB containing 50 µg/ml ampicillin.•Grow the culture to mid-log phase (OD600 = 0.5-0.7).•Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial.•Store at -80°C.TransfectionIntroduction Once you have verified that your gene is cloned in the correct orientation and contains an initiation ATG and a stop codon, you are ready to transfect your cell line of choice. Werecommend that you include the positive control vector and a mock transfection (negativecontrol) to evaluate your results.Plasmid Preparation Plasmid DNA for transfection into eukaryotic cells must be clean and free from phenol and sodium chloride. Contaminants will kill the cells, and salt will interfere with lipids decreasing transfection efficiency. We recommend isolating plasmid DNA using the S.N.A.P.™ MiniPrep Kit (10-15 µg DNA, Catalog no. K1900-01), the S.N.A.P. ™MidiPrep Kit (10-200 µg DNA, Catalog no. K1910-01), or CsCl gradient centrifugation.Methods of Transfection For established cell lines (e.g. HeLa), please consult original references or the supplier of your cell line for the optimal method of transfection. We recommend that you follow exactly the protocol for your cell line. Pay particular attention to medium requirements, when to pass the cells, and at what dilution to split the cells. Further information is provided in Current Protocols in Molecular Biology (Ausubel et al., 1994).Methods for transfection include calcium phosphate (Chen and Okayama, 1987; Wigler et al., 1977), lipid-mediated (Felgner et al., 1989; Felgner and Ringold, 1989) and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988). Invitrogen offers the Calcium Phosphate Transfection Kit (Catalog no. K2780-01) and a large selection of reagents for transfection. For more information, please refer to our World Wide Web site () or call Technical Service (see page 13).Positive Control pcDNA3.1/CAT is provided as a positive control vector for mammalian transfection and expression (see page 12) and may be used to optimize transfection conditions for yourcell line. The gene encoding chloramphenicol acetyl transferase (CAT) is expressed inmammalian cells under the control of the CMV promoter. A successful transfection willresult in CAT expression that can be easily assayed (see below).Assay for CAT Protein You may assay for CAT expression by ELISA assay, western blot analysis, fluorometric assay, or radioactive assay (Ausubel et al., 1994; Neumann et al., 1987). If you wish to detect CAT protein using western blot analysis, you may use the Anti-CAT Antiserum (Catalog no. R902-25) available from Invitrogen. Other kits to assay for CAT protein using ELISA assay are available from Roche Molecular Biochemicals (Catalog no. 1 363 727) and Molecular Probes (Catalog no. F-2900).Creation of Stable Cell LinesIntroduction The pcDNA3.1(+) and pcDNA3.1(-) vectors contain the neomycin resistance gene forselection of stable cell lines using neomycin (Geneticin®). We recommend that you testthe sensitivity of your mammalian host cell to Geneticin® as natural resistance variesamong cell lines. General information and guidelines are provided in this section for yourconvenience.Geneticin®Selective Antibiotic Geneticin® Selective Antibiotic blocks protein synthesis in mammalian cells by interfering with ribosomal function. It is an aminoglycoside, similar in structure to neomycin, gentamycin, and kanamycin. Expression of the bacterial aminoglycoside phosphotransferase gene (APH), derived from Tn5, in mammalian cells results in detoxification of Geneticin®(Southern and Berg, 1982).Geneticin®Selection Guidelines Geneticin® Selective Antibiotic is available from Invitrogen (Catalog no. 10486-025). Use as follows:•Prepare Geneticin® in a buffered solution (e.g. 100 mM HEPES, pH 7.3).•Use 100 to 800 µg/ml of Geneticin® in complete medium.•Calculate concentration based on the amount of active drug (check the lot label).•Test varying concentrations of Geneticin® on your cell line to determine theconcentration that kills your cells (see below). Cells differ in their susceptibility to Geneticin®.Cells will divide once or twice in the presence of lethal doses of Geneticin®, so the effects of the drug take several days to become apparent. Complete selection can take up to 3 weeks of growth in selective media.Determination of Antibiotic Sensitivity To successfully generate a stable cell line expressing your gene of interest frompcDNA3.1, you need to determine the minimum concentration of Geneticin® required to kill your untransfected host cell line. We recommend that you test a range of concentrations to ensure that you determine the minimum concentration necessary for your host cell line.1.Plate or split a confluent plate so the cells will be approximately 25% confluent.Prepare a set of 7 plates. Allow cells to adhere overnight.2.The next day, substitute culture medium with medium containing varyingconcentrations of Geneticin® (0, 50, 100, 200, 400, 600, 800 µg/ml Geneticin®).3.Replenish the selective media every 3-4 days, and observe the percentage of survivingcells.4.Count the number of viable cells at regular intervals to determine the appropriateconcentration of Geneticin® that prevents growth within 2-3 weeks after addition of Geneticin®.continued on next pagePossible Sites for Linearization of pcDNA3.1(+)Prior to transfection, we recommend that you linearize the pcDNA3.1(+) vector. Linearizing pcDNA3.1(+) will decrease the likelihood of the vector integrating into the genome in a way that disrupts the gene of interest or other elements required for expression in mammalian cells. The table below lists unique restriction sites that may be used to linearize your construct prior to transfection. Other unique restriction sites are possible. Be sure that your insert does not contain the restriction enzyme site you wish to use to linearize your vector.Enzyme Restriction Site (bp)Location SupplierBgl II12Upstream of CMV promoter Invitrogen, Catalog no. 15213-028 Mfe I161Upstream of CMV promoter New England BiolabsBst1107 I3236End of SV40 polyA AGS*, Fermentas, Takara, RocheMol. BiochemicalsEam1105 I4505Ampicillin gene AGS*, Fermentas, TakaraPvu I4875Ampicillin gene Invitrogen, Catalog no. 25420-019 Sca I4985Ampicillin gene Invitrogen, Catalog no. 15436-017 Ssp I5309bla promoter Invitrogen, Catalog no. 15458-011 *Angewandte Gentechnologie SystemePossible Sites for Linearization of pcDNA3.1(-)The table below lists unique restriction sites that may be used to linearize yourpcDNA3.1(-) construct prior to transfection. Other unique restriction sites are possible. Be sure that your insert does not contain the restriction enzyme site you wish to use to linearize your vector.Enzyme Restriction Site (bp)Location SupplierBgl II12Upstream of CMV promoter Invitrogen, Catalog no. 15213-028 Mfe I161Upstream of CMV promoter New England BiolabsBst1107 I3235End of SV40 polyA AGS*, Fermentas, Takara, RocheMol. BiochemicalsEam1105 I4504Ampicillin gene AGS*, Fermentas, TakaraPvu I4874Ampicillin gene Invitrogen, Catalog no. 25420-019 Sca I4984Ampicillin gene Invitrogen, Catalog no. 15436-017 Ssp I5308bla promoter Invitrogen, Catalog no. 15458-011 *Angewandte Gentechnologie Systemecontinued on next pageSelection of Stable Integrants Once you have determined the appropriate Geneticin® concentration to use for selection in your host cell line, you can generate a stable cell line expressing your gene of interest.1.Transfect your mammalian host cell line with your pcDNA3.1 construct using thedesired protocol. Remember to include a plate of untransfected cells as a negativecontrol and the pcDNA3.1/CAT plasmid as a positive control.2.24 hours after transfection, wash the cells and add fresh medium to the cells.3.48 hours after transfection, split the cells into fresh medium containing Geneticin® atthe pre-determined concentration required for your cell line. Split the cells such that they are no more than 25% confluent.4.Feed the cells with selective medium every 3-4 days until Geneticin®-resistant foci canbe identified.5.Pick and expand colonies in 96- or 48-well plates.AppendixpcDNA3.1 VectorsMap ofpcDNA3.1(+) and pcDNA3.1(-)The figure below summarizes the features of the pcDNA3.1(+) and pcDNA3.1(-) vectors.The complete sequences for pcDNA3.1(+) and pcDNA3.1(-) are available for down-loading from our World Wide Web site ( ) or from Technical Service (see page 13). Details of the multiple cloning sites are shown on page 3 for pcDNA3.1(+) and page 4 for pcDNA3.1(-).Comments for pcDNA3.1 (+) 5428 nucleotidesCMV promoter: bases 232-819T7 promoter/priming site: bases 863-882Multiple cloning site: bases 895-1010BGH polyadenylation sequence: bases 1028-1252f1 origin: bases 1298-1726SV40 early promoter and origin: bases 1731-2074Neomycin resistance gene (ORF): bases 2136-2930SV40 early polyadenylation signal: bases 3104-3234pUC origin: bases 3617-4287 (complementary strand)Ampicillin resistance gene (bla ): bases 4432-5428 (complementary strand) ORF: bases 4432-5292 (complementary strand)Ribosome binding site: bases 5300-5304 (complementary strand) bla promoter (P3): bases 5327-5333 (complementary strand)I I (+)( )continued on next pagepcDNA3.1 Vectors, continuedFeatures of pcDNA3.1(+) and pcDNA3.1(-)pcDNA3.1(+) (5428 bp) and pcDNA3.1(-) (5427 bp) contain the following elements. All features have been functionally tested.Feature BenefitHuman cytomegalovirus (CMV)immediate-early promoter/enhancerPermits efficient, high-level expression ofyour recombinant protein (Andersson et al.,1989; Boshart et al., 1985; Nelson et al.,1987)T7 promoter/priming site Allows for in vitro transcription in the senseorientation and sequencing through theinsertMultiple cloning site in forward orreverse orientationAllows insertion of your gene andfacilitates cloningBovine growth hormone (BGH)polyadenylation signalEfficient transcription termination andpolyadenylation of mRNA (Goodwin andRottman, 1992)f1 origin Allows rescue of single-stranded DNASV40 early promoter and origin Allows efficient, high-level expression ofthe neomycin resistance gene and episomalreplication in cells expressing SV40 large TantigenNeomycin resistance gene Selection of stable transfectants inmammalian cells (Southern and Berg,1982)SV40 early polyadenylation signal Efficient transcription termination andpolyadenylation of mRNApUC origin High-copy number replication and growthin E. coliAmpicillin resistance gene (β-lactamase)Selection of vector in E. colipcDNA3.1/CATDescription pcDNA3.1/CAT is a 6217 bp control vector containing the gene for CAT. It wasconstructed by digesting pcDNA3.1(+) with Xho I and Xba I and treating with Klenow.An 800 bp Hin d III fragment containing the CAT gene was treated with Klenow and thenligated into pcDNA3.1(+).Map of Control Vector The figure below summarizes the features of the pcDNA3.1/CAT vector. The complete nucleotide sequence for pcDNA3.1/CAT is available for downloading from our World Wide Web site () or by contacting Technical Service (see page 13).Comments for pcDNA3.1(+)/CAT6217 nucleotidesCMV promoter: bases 232-819CAT ORF: bases 1027-1686pcDNA3.1/BGH reverse priming site: bases 1811-1828BGH polyadenylation sequence: bases 1817-2041f1 origin: bases 2087-2515SV40 early promoter and origin: bases 2520-2863Neomycin resistance gene (ORF): bases 2925-3719SV40 early polyadenylation sequence: bases 3893-4023pUC origin: bases 4406-5076 (complementary strand)Ampicillin resistance gene (ORF): bases 5221-6081 (complementary strand)Technical ServiceVisit the Invitrogen Web Resource using your World Wide Web browser. At the site, youcan:•Get the scoop on our hot new products and special product offers•View and download vector maps and sequences•Download manuals in Adobe® Acrobat® (PDF) format•Explore our catalog with full color graphics•Obtain citations for Invitrogen products•Request catalog and product literatureOnce connected to the Internet, launch your web browser (Internet Explorer 5.0 or neweror Netscape 4.0 or newer), then enter the following location (or URL):...and the program will connect directly. Click on underlined text or outlined graphics toexplore. Don't forget to put a bookmark at our site for easy reference!Contact us For more information or technical assistance, please call, write, fax, or email. Additionalinternational offices are listed on our web page ().United States Headquarters:Japanese Headquarters European Headquarters:Invitrogen Corporation Invitrogen Japan K.K.Invitrogen Ltd1600 Faraday Avenue Nihonbashi Hama-Cho Park Bldg. 4F 3 Fountain DriveCarlsbad, CA 92008 USA2-35-4, Hama-Cho, Nihonbashi Inchinnan Business ParkTel: 1 760 603 7200Tel: 81 3 3663 7972Paisley PA4 9RF, UKTel (Toll Free): 1 800 955 6288Fax: 81 3 3663 8242Tel (Free Phone Orders): 0800 269 210 Fax: 1 760 602 6500E-mail: jpinfo@ Tel (General Enquiries): 0800 5345 5345 E-mail:Fax: +44 (0) 141 814 6287tech_service@ E-mail: eurotech@MSDS Requests To request an MSDS, please visit our web site () and follow theinstructions below.1.On the home page, go to the left-hand column under ‘Technical Resources’ andselect ‘MSDS Requests’.2.Follow instructions on the page and fill out all the required fields.3.To request additional MSDSs, click the ‘Add Another’ button.4.All requests will be faxed unless another method is selected.5.When you are finished entering information, click the ‘Submit’ button. Your MSDSwill be sent within 24 hours.continued on next pageTechnical Service, continuedEmergency Information In the event of an emergency, customers of Invitrogen can call the 3E Company, 24 hours a day, 7 days a week for disposal or spill information. The 3E Company can also connect the customer with poison control or with the University of California at San Diego Medical Center doctors.3E CompanyVoice: 1-760-602-8700Limited Warranty Invitrogen is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you shouldhave any questions or concerns about an Invitrogen product or service, please contact ourTechnical Service Representatives.Invitrogen warrants that all of its products will perform according to the specifications stated on thecertificate of analysis. The company will replace, free of charge, any product that does not meetthose specifications. This warranty limits Invitrogen Corporation’s liability only to the cost of theproduct. No warranty is granted for products beyond their listed expiration date. No warranty isapplicable unless all product components are stored in accordance with instructions. Invitrogenreserves the right to select the method(s) used to analyze a product unless Invitrogen agrees to aspecified method in writing prior to acceptance of the order.Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that theoccasional typographical or other error is inevitable. Therefore Invitrogen makes no warranty ofany kind regarding the contents of any publications or documentation. If you discover an error inany of our publications, please report it to our Technical Service Representatives.Invitrogen assumes no responsibility or liability for any special, incidental, indirect orconsequential loss or damage whatsoever. The above limited warranty is sole and exclusive.No other warranty is made, whether expressed or implied, including any warranty ofmerchantability or fitness for a particular purpose.ReferencesAndersson, S., Davis, D. L., Dahlbäck, H., Jörnvall, H., and Russell, D. W. (1989). Cloning, Structure, andExpression of the Mitochondrial Cytochrome P-450 Sterol 26-Hydroxylase, a Bile Acid Biosynthetic Enzyme. J.Biol. Chem. 264, 8222-8229.Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994).Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience).Boshart, M., Weber, F., Jahn, G., Dorsch-Häsler, K., Fleckenstein, B., and Schaffner, W. (1985). A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus. Cell 41, 521-530.Chen, C., and Okayama, H. (1987). High-Efficiency Transformation of Mammalian Cells by Plasmid DNA. Mol.Cell. Biol. 7, 2745-2752.Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the Efficient Transfection of Mammalian Cells with DNA. Nuc. Acids Res. 15, 1311-1326.Felgner, P. L., Holm, M., and Chan, H. (1989). Cationic Liposome Mediated Transfection. Proc. West. Pharmacol.Soc. 32, 115-121.Felgner, P. L., and Ringold, G. M. (1989). Cationic Liposome-Mediated Transfection. Nature 337, 387-388.Goodwin, E. C., and Rottman, F. M. (1992). The 3´-Flanking Sequence of the Bovine Growth Hormone GeneContains Novel Elements Required for Efficient and Accurate Polyadenylation. J. Biol. Chem. 267, 16330-16334. Kozak, M. (1987). An Analysis of 5´-Noncoding Sequences from 699 Vertebrate Messenger RNAs. Nuc. Acids Res.15, 8125-8148.Kozak, M. (1991). An Analysis of Vertebrate mRNA Sequences: Intimations of Translational Control. J. Cell Biol.115, 887-903.Kozak, M. (1990). Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes. Proc. Natl. Acad. Sci. USA 87, 8301-8305.Nelson, J. A., Reynolds-Kohler, C., and Smith, B. A. (1987). Negative and Positive Regulation by a Short Segmentin the 5´-Flanking Region of the Human Cytomegalovirus Major Immediate-Early Gene. Mol. Cell. Biol. 7, 4125-4129.Neumann, J. R., Morency, C. A., and Russian, K. O. (1987). A Novel Rapid Assay for Chloramphenicol Acetyltransferase Gene Expression. BioTechniques 5, 444-447.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).Shigekawa, K., and Dower, W. J. (1988). Electroporation of Eukaryotes and Prokaryotes: A General Approach tothe Introduction of Macromolecules into Cells. BioTechniques 6, 742-751.Southern, P. J., and Berg, P. (1982). Transformation of Mammalian Cells to Antibiotic Resistance with a BacterialGene Under Control of the SV40 Early Region Promoter. J. Molec. Appl. Gen. 1, 327-339.Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y.-C., and Axel, R. (1977). Transfer of Purified HerpesVirus Thymidine Kinase Gene to Cultured Mouse Cells. Cell 11, 223-232.©1997-2001 Invitrogen Corporation. All rights reserved.15。

质粒图谱查询方法

3.google scholar: / 有些质粒是经过改造的，所以通过上述方法不能查询到相应信息。这时，可以在google scholar中输入质粒名称，可以直观地看哪些学者在何文章中使用了该质粒，从而可了解到质粒的来源;或者籍此向作者咨询或索取。 4.尝试从各大生物公司，例如invitrogen网站查询. 5. 这个网站收录了大量图谱： http://www.embl-hamburg.de/~geerlof/webPP/vectordb/bact_vectors/table.html
By
0039--pET-24a--Novagene--原核表达--yanBaggio 0040--pGBKT7 和pGADT7--CLOTE--酵母表达--小迷糊 0041--PTXB1--BIOLABS--原核表达--小迷糊 0042--pET-43.1 Ek/LIC--Novagene--原核表达--mcli 0043--PIRES--BD BIOSCIENCES--穿梭载体---joeys008 0044--pBAD/Thio-TOPO--invitrogen--原核表达--mcli 0045--pESC-HIS--Stratagene--酵母表达--zhangqiongyu82 0046--pcDNA3.1/V5-His-TOPO--invitrogen--真核表达--mcli 0047--pMSCVneo--Clontech--真核表达--intron 0048--pCDNA6 /V5 HisB--invitrogen--真核表达--linct97 0049--pcDNA3.1(+)--invitrogen--真核表达--kinase 0050--pSecTag2--invitrogen--真核表达--sssusu 0051--pIRESneo--Clontech--真核表达--sssusu 0052--pBudCE4.1--invitrogen--真核表达--barbie 0053--pGEX-4T-3--amershambio--原核表达--linct97 0054--PESC-TRP--startgene--真核表达--zhangqiongyu82 0055--pPICZ--invitrogen--毕赤酵母表达--yshu3507 0056--pTrc99a--novogen--原核表达--Gmail 0057--pGEX-6p-1--amershambio--原核表达--erik 0058--pSFV1--invitrogen--真核表达--syfnet 0059--pSCA1--赠送--真核表达--syfnet 0060--pUC18--/--克隆--syfnet 0061--pshuttleCMV--KRACKELER--穿梭质粒--renke3333 0062--pdc315--microbix--shuttle--renke3333 0063--pBluescript SK(+)--tianwei--原核表达--fenqinzhuhe 0064--pPIC3.5K--Invitrogen--真核表达--mcli 0065--pFB-hrGFP--Stratagene--真核表达--zhangqiongyu82 0066--pSG5--Stratagene--真核表达--zhangqiongyu82 0067--pCMV-Tag4B--Stratagene--真核表达--小迷糊 0068--pET11a--invitrogen--原核表达--pinghw 0069--pET30a--invitrogen--原核表达--seasider 0070--pCI-NEO--PREMOGA--真核表达--syfnet 0071--phRL-null--PREMOGA--真核表达--kenmed 0072--pBC1--invitrogen--真核表达--竹影烟雨 0073--pGEMEX-1--Promega--体外转录--palmyard 0074--pGEM-T Easy--Promega--克隆--laohu200381 0075--pGEX-5x-1--Pharmacia--原核表达--laohu200381 0076--pshuttle--qbiogene--穿梭质粒--renke3333 0077--pet9c--promega--原核表达--renke3333 0078--trans-vector--qbiogene--穿梭质粒--renke3333 0079--PQBI PGK--QBIOGENE--真核表达--renke3333 0080--PQBI T7 GFP--QBIOGENE--真核表达--renke3333 0081--pcDNA5/FRT/CAT—INVITROGEN--真核表达--renke3333 0082--pBABE Hygro—Geron--真核表达--Jeffrey88 0083--pBABE puro--Geron--Jeffrey88 0084--pMSCV puro—Clontech—RNAi--tuterhu 0085--质粒名--PEGFPN3—Clontech--真核 GFP--renke3333 0086--质粒名称—PSHTULLE—Clontech--穿梭---renke3333 0087--pkk223-3--哈佛大学Brosius等构建--原核表达--kingtsh 0088--pcdna3-c-myc—invitroge--真核表达--giyon 0089—pSG-cmv--新基因--stevenvin 0090--PGEX-3x--BD Co--融合型蛋白原核表达载体--kingtsh 0091--PEGFPc2—Clontech--真核 GFP 0092--PEGFP－N2—Clontech--真核 GFP 0093—PMECA--不详--克隆载体 0094--PGEX 4T－2--不详--克隆载体 0095--siSTRIKE? U6—PROMEGA--RNAi载体 0096--pSilencer? neo—Ambion--RNAi载体 0097--pSilencer? hygro—Ambion--RNAi载体 0098--pSilencer? puro—Ambion--RNAi载体

质粒图谱——精选推荐

1)质粒图谱登记号：00012)质粒名称：pIRES3)来源：BD Co4)用途：真核双表达5)是否可以提供更详细资料：可以6)是否可以共享：7)联系方式：PM)质粒图谱登记号：00022)质粒名称：pECFP-C13)来源：BD Co4)用途：检测真核表达5)是否可以提供更详细资料：可以6)是否可以共享：7)联系方式：pm1)质粒图谱登记号：00032)质粒名称：pShuttle3)来源：4)用途：5)是否可以提供更详细资料：6)是否可以共享：7)联系方式：2)pShuttle MCS很多人用pEGFP-C1，我也来发一个)质粒图谱登记号：00042)质粒名称：pS BR322、pUC183)来源：4)用途：5)是否可以提供更详细资料：6)是否可以共享：7)联系方式：1)质粒图谱登记号：00052)质粒名称：pcDNA3.1(+)/CAT3)来源：invitrogen4)用途：真核表达5)是否可以提供更详细资料：可以6)是否可以共享：无偿7)联系方式：PM1)质粒图谱登记号：00062)质粒名称：pQEx3)来源：Qiagen4)用途：原核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM2)1)质粒图谱登记号：00072)质粒名称：pIVEX2.33)来源：Rocho4)用途：体外转录翻译5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM质粒图谱登记号：0008质粒名称：pIRES-EGFP来源：用途：是否可以提供更详细资料：是否可以共享：否联系方式：1)质粒图谱登记号：00092)质粒名称：pET-28a（+）3)来源：Novagen4)用途：原核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM1)质粒图谱登记号：00112)质粒名称：pET-32a(+)3)来源：novagen4)用途：原核表达5)是否可以提供更详细资料：6)是否可以共享：7)联系方式：pm1)质粒图谱登记号：00132)质粒名称：pcDNA3.1/Zeo (+)3)来源：invitrogen4)用途：原核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM)质粒图谱登记号：00132)质粒名称：pEGFP-N33)来源：clontech4)用途：真核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM)质粒图谱登记号：00142)质粒名称：pcDNA33)来源：invitrogen4)用途：真核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM)质粒图谱登记号：00152)质粒名称：pfastbac13)来源：invitrogene4)用途：昆虫表达5)是否可以提供更详细资料：不可以6)是否可以共享：不可以7)联系方式：PM)质粒图谱登记号：00162)质粒名称：pEGFP-C33)来源：clontech4)用途：真核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PMwangjun2002274 edited on 2004-06-22 00:041)质粒图谱登记号：00172)质粒名称：pSecTag23)来源：Invitrogen4)用途：真核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM)1质粒图谱登记号：00192)质粒名称：pET20b3)来源：NOVAGEN4)用途：原核表达5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM1)质粒图谱登记号：00222)质粒名称：pThioHisA3)来源：invitrogen4)用途：原核表达5)是否可以提供更详细资料：4.365kb , HP-thioredoxin fusion proteinexpressionvector, trc promoter, Ampr, a EK cleavage site lies between HP-thioredoxin and MCS宿主菌TOP10（基因型为：F-mcrA △（mrr-hsd RMS-mcrBC）Ф80 lacZ M15 △lacX74 deoR recAl araD139 △（ara-leu）7697 galU galK rpsL endAl nupG6)是否可以共享：交换或其它7)联系方式：PM2)3)1)质粒图谱登记号：00242)质粒名称：pcDNA3.1-Myc-His-A-3)来源：invitrogen4)用途：真核核表达4))质粒图谱登记号：00252)质粒名称：pS UPER.neo3)来源：4)用途：siRNA5)是否可以提供更详细资料：可以6)是否可以共享：交换7)联系方式：PM)质粒图谱登记号：00312)质粒名称：pSilencer1.0-siRNA3)来源：Ambion4)用途：RNAi5)是否可以提供更详细资料：/techlib/Documents.html?fkResSxn=7&fkSub Sxn=236)是否可以共享：实验结束后，可提供含shRNA模板的质粒7)联系方式：pm5))质粒图谱登记号：00322)质粒名称：pSilencer2.0-U6siRNA3)来源：Ambion4)用途：RNAi，与1.0相比，可以建立稳转株5)更详细资料：/techlib/prot/fm_7209.pdf6)是否可以共享：交换7)联系方式：pm6)会员名：mlluoE-mail：*************可提供试验资源名称和简要介绍：pSilencer 3.1-H1 neo Vector，是Ambion公司目前最高版本的shRNA 载体，为扩增此载体我已插入目的片段，如有战友需要，将此片段双酶切再连上自己的片段即可。

各类质粒载体图谱

(PR8Z151)
pGADT7
Vector Information
as a fusion to a hemagglutinin (HA) epitope tag. HA-tagged proteins can be identified with antibodies raised to this common epitope, eliminating the need to generate specific antibodies to new proteins. The T7 promoter is used for in vitro transcription and translation of the epitope tagged fusion protein and also provides a binding site for sequencing using the T7 Sequencing Primer. Note that the AD is not expressed during the in vitro transcription and translation reactions. The Nco I and Pst I sites may be used to shuttle inserts from pGADT7 into pGBKT7, the MATCHMAKER Two-Hybrid System 3 DNA-BD Vector. The MCS in pGADT7 is compatible with those in pMyc-CMV and pHA-CMV, CLONTECH's epitope tagged mammalian expression vector set (#K6003-1). As a result, the target gene can be shuttled into these vectors in order to confirm protein interactions in vivo. Location of features: • Full-length S. cerevisiae ADH1 promoter (PADH1): 7–1479 • GAL4 AD polypeptide with SV40 Nuclear Localization Signal (NLS) NLS: 1501–1557 GAL4 amino acids 768–881: 1561–1899 • T7 RNA polymerase promoter: 1905–1927 • HA epitope tag: 1942–1968 • Multiple Cloning Sites: 1969–2041 • Transcription termination signal Fragment carrying the S. cerevisiae ADH1 terminator (TADH1): 2280–2605 • LEU2 coding sequences: 3814–2723 • pUC plasmid replication origin: 4581–5418 • Ampicillin resistance gene: 6432–5575 • Yeast 2 µ replication origin: 6998–7988 Location of primers: • T7 Sequencing Primer: 1905–1925 • 3' AD Sequencing Primer: 2102–2083 • MATCHMAKER 5' AD LD-Insert Screening Amplimer (#9103-1): 1858–1889 • MATCHMAKER 3' AD LD-Insert Screening Amplimer (#9103-1): 2078–2046 Propagation in E. coli: • Suitable host strains: DH5α, DH10 & other general purpose strains • Selectable marker: plasmid confers resistance to ampicillin (100 µg/ml) to E. coli hosts • E. coli replication origin: pUC • Copy number: ~500 • Plasmid incompatibility group: pMB1/Col E1 Propagation in S. cerevisiae: • Suitable host strains: Y187(α), Y190(a), SFY526(a), CG1945(a), HF7c(a), or AH109(a) • Selectable marker: LEU2 • S. cerevisiae origin: 2 µ Reference:

pmd18-19-T 质粒图谱

pMD18-T or pMD19-T Vector*1 Insert DNA*3 dH2O
1 µl 0.1 pmol ~ 0.3 pmol
up to 5 µl
2）加入5 µl（等量）的 Ligation Mix。 3）16℃反应30分钟。注）① 室温（25℃）也能正常进行连接反应，但反应
效率稍微降低。 ② 5分钟也能正常进行连接反应，但反应效率稍微
α多肽相结
合，表现出 β-半乳糖苷酶活性（α-互补性)。
2. Insert DNA的要求。
Insert DNA应该进行切胶回收的纯化处理后才进行载体
连接，尽量避免引物等其它杂质的存在。切胶回收时
可使用TaKaRa凝胶回收试剂盒（TaKaRa Code：D301
或DV805A）。
3. Insert DNA使用量的计算方法。
dna片段成功插入至pmd18?tvectorpmd19?tvector中后一般情况下?半乳糖苷酶的表达将受到破坏重组克隆体在含有x?galiptgamp的l?琼脂平板培养基上培养时将显示白色菌?
PCR产物克隆系列载体
Amp r
pMD18-T Vector pMD19-T Vector
D101A D102A
BcaBESTTM Sequencing Primer RV-M
GAGCGGATAACAATTTCACACAGG lacZ
Hind III
Hinc II Sse8387 I Acc I
Sph I Pst I Sal I EcoR V
Xba I
BamH I
Xma I Sma I Kpn I
Sac I
EcoR I
较短的DNA片段插入载体时，基因的读码框有可能正

pet32a质粒图谱

pet32a质粒图谱pet32a质粒是一种重要的研究工具，用于分子生物学相关实验中的蛋白质表达和纯化。

它是一种广泛应用的质粒，具有较高的表达效率和纯化便利性。

本文将介绍pet32a质粒的基本信息、构建原理以及它在分子生物学研究中的应用。

基本信息pet32a质粒是一种循环DNA分子，含有多个重要元素，包括起始子、转录终止子、选择标记、操纵子等。

它的大小为5406bp，可以轻松通过现有的克隆技术进行构建和改造。

pet32a质粒的选择标记是T7表达子，该序列在大肠杆菌中能够高效表达外源蛋白。

此外，pet32a质粒还具有His_6标记，便于后续纯化步骤。

这些特征使得pet32a质粒成为常用的表达载体。

构建原理pet32a质粒的构建原理基于DNA重组技术，通过将所需的基因序列插入质粒中的多个限制性内切酶位点来实现。

通常，选择一个合适的限制性内切酶对来线性化pet32a质粒，然后将目标基因片段与线性化的质粒进行连接。

最后，将重组后的质粒转化到宿主细胞中，进行筛选、培养和表达。

在构建过程中，需要注意以下几个关键点。

1.合适的限制性内切酶选择：根据目标基因片段的序列选择适合的限制性内切酶，确保酶切位点互不干扰。

2.合成适当的引物：为目标基因片段的插入和PCR扩增合成合适的引物，确保插入序列的准确性和完整性。

3.质粒与目标基因片段的平衡：在重组过程中控制质粒与目标基因片段的比例和浓度，避免过多或过少的插入，影响质粒的稳定和表达效率。

应用pet32a质粒广泛应用于分子生物学研究中的蛋白质表达和纯化。

对于蛋白质表达，pet32a质粒基于T7表达子高效表达外源蛋白，为蛋白质结构和功能研究提供了可靠的工具。

利用pet32a质粒，研究者可以通过蛋白质表达和纯化流程获取大量高纯度的目标蛋白。

这对于结构生物学、药物研发等领域的研究具有重要意义。

在纯化过程中，pet32a质粒具有His_6标记，可通过亲和层析技术高效纯化目标蛋白。