常用pGEX载体图谱
pGEX-4T-1
Version 03302011 pGEX-4T-1 VectorspGEX-4T-1 载体Cat. No. CW2198保存：-20℃组分说明Cat. No. CW2198pGEX4T-1 Vectors 20 μl（100 ng/μl）产品简介pGEX-4T-1载体是一种常用的原核表达载体，具有Amp抗性，质粒大小为4.9 kb。
该载体多克隆位点区域含有多个常用的内切酶位点序列，便于不同基因的克隆；且全序列中含有tac启动子、GST标签序列，是一种高效的蛋白表达载体。
Tac 启动子在没有诱导物存在的情况下，质粒上携带的lacI q基因产物可以有效抑制tac启动子的转录。
可在BL21、Rosstea等表达菌株中高表达融合有GST标签的蛋白，且用凝血酶可将目的蛋白的GST标签切掉，便于下游蛋白纯化。
载体图谱及多克隆位点附表 标记序列位点标记序列 位点AmpR_promotor1307-1335 Orf2 3441-4400 Orf2 258-977 pGEX_5_primer 869-891 GST 258-977 tac_promotor 184-212 Orf2 1377-2237 lacI 3309-4400 lac_promotor 4449-4478 M13_forward20_primer4559-4543 lacZ_a 4540-4695 M13_reverse_primer4513-4531 M13_pUC_fwd_primer 4574-4552 M13_pUC_rev_primer 224-246 Ampicilin 1377-2237 pGEX_3_primer 1041-1019 Pbr322_origin 2392-3011 M13_pUC_rev_primer 4492-4514。
质粒图谱大全
〔转载〕Pllp-OmpA, pllp-STII, pMBP-P, pMBP-C,pET-GST, pET-Trx, pET-His, pET-CKS, pET-DsbApTZ19RDNApUC57DNAPMD18TPQE30pUC18pUC19pTrcHisApTrxFuspRSET-ApRSET-BpVAX1PBR322pbv220pBluescriptIIKS( )L4440pCAMBIA-1301pMAL-p2XpGD926ProteinExpression?ProkaryoticExpression?pETDsbFusionSystems39band40b ProteinExpression?ProkaryoticExpression?pETExpressionSystem33b ProteinExpression?ProkaryoticExpression?pETExpressionSystems ProteinExpression?ProkaryoticExpression?pETExpressionSystemsplusCompetentCells ProteinExpression?ProkaryoticExpression?pETGSTFusionSystems41and42 ProteinExpression?ProkaryoticExpression?pETVectorDNAProteinPurification?PurificationSystems?Strep?TactinResinsandPurificationKitsTEcoR?pGEX-1I/BAPpGEX-2TpGEX-2TKpGEX-3XpGEX-4T-1pGEX-4T-2pGEX-4T-3pGEX-5X-1pGEX-5X-2pGEX-5X-3pGEX-6P-1pGEX-6P-2pGEX-6P-3PTYB1PTYB2PTYB11PTYB12pCDNA3.1(-)pCDNA3.1( )pPICZalphaApGAPZαApBI121pEGFP-N1pEGFP-C1pPIC9K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。
载体图谱
一、pGADT7(Amp)酵母表达载体(AD-Amp)AD forward primer: 5'-TAATACGACTCACTATAGGGC-3'AD reverse primer: 5'-AGATGGTGCACGATGCACAG-3'F：GC AT CGA TAC ATG GC T TG T GCTACTCTGAR：GGCC GGATCC TTAAGAGAGGTAGCTGGGAGAD forward primer: 5'-TAATACGACTCACTATAGGGC-3'AD reverse primer: 5'-AGATGGTGCACGATGCACAG-3'二、pGBKT7(Kan0酵母表达载体(BD-Kan)三、BIFC(Kan)验证互做蛋白表达载体（德国）四、pGR107(Kan)表达载体（PVX-10kb）吴建国MCS:ClaI/SmaI/SalI(pgR107)MCS:ClaI/AscI/NotI/SalI(pgR106) 五、BIFC(Amp)载体（美国）3075（nEYFP-329bp）3076 (cEYFP-218bp)F：GGC GAATTC ATG GCTTGTGCTACTCTGA R：GGC CCTAGG AGAGAGGTAGCTGGGAG TAA TAG TGA六、RNAI(Kan)载体-pFGC5941（王宗华）七、pGEX-4T-1(Amp)原核表达载体八：pET-29a(Kan)原核表达载体九、. pEGAD(Kan)植物表达载体*XbaⅠ cleavage blocked by overlapping dam methylation. Must grow in dam-strain to cut. Note, XhoⅠ is not unique.pTRV2(a) Genome organization of TRV. The TRV-RNA1 open reading frames (ORFs) correspond to 134 and 194 kDa replicase, movement protein (MP) and a 16-kDa cysteine-rich protein. The TRV-RNA2 ORFs corresponds to coat protein (CP), and the 29.4 and 32.8 kDa proteins. gRNA, genomic RNA; sgRNA, subgenomic RNA. Asterisks (*) indicate the readthrough of 134 kDa protein.(b) TRV based VIGS vectors. TRV cDNA clones were placed in betweenthe duplicated CaMV 35S promoter (2×35S) and the nopaline synthase terminator (NOSt) in a T-DNA vector. LB and RB refer to left and right borders of T-DNA. Rz, self-cleaving ribozyme. MCS, multiple cloning sites.十、十一、1103-YFP十二：pBINPLUS十三、pBin438。
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。
pGEX质粒图谱
pGEX-1λT (27-4805-01)pGEX-6P-1 (27-4597-01)EcoR I Sma I Sal I Xho I Not IBamH I PreScission ™ ProteaseLeu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro HisCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT↓pGEX-6P-2 (27-4598-01)BamH I PreScission ™ ProteaseLeu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG↓EcoR I Sal I Xho INot I pGEX-6P-3 (27-4599-01)BamH IPreScission ™ ProteaseLeu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly ArgCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC↓EcoR I Sma I Sal I Xho I Not I BamH IEcoR I Sma I Sal I Xho I Not I BamH I EcoR I Sma I Sal I Xho I Not IBamH I EcoR I Sma I Sal I Xho I Not IBamH I EcoR I Sma I Sal I Xho I Not IBamH I EcoR I Sma I Sal I Xho INot I BamH I EcoR I Sma I Sal I Not IEcoR I CTG GTT CCG CGT GGA TCC CCG GAA TTC ATC GTG ACT GAC TGA CGABamH ILeu Val Pro Arg Gly Ser Pro Glu Phe Ile Val Thr AspThrombinStop codons ↓pGEX~4900 bppBR322oriBal I BspM IPtac g lu ta thi on e S -t r a n s f e r as eA mp ra c Iq Nar I EcoR VBssH IIBstE IIMlu IApa ITth111 IAat IIPst Ip4.5AlwN IpSj10 Bam7Stop7∆pGEX-4T-2 (27-4581-01)pGEX-5X-1 (27-4584-01)pGEX-5X-2 (27-4585-01)pGEX-5X-3 (27-4586-01)pGEX-4T-1 (27-4580-01)pGEX-4T-3 (27-4583-01)pGEX-3X (27-4803-01)pGEX-2TK (27-4587-01)Leu Val Pr o Ar g Gly Ser Pr o Gly Ile Pr o Gly Ser Thr Ar g Ala Ala Ala SerCTG GTT CCG CGT GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGAStop codon Ile Glu Gly Arg Gly Ile Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg AspATC GAA GGT CGT GGG ATC CCC GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGAStop codons Ile Glu Gly Arg Gly Ile Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerATC GAA GGT CGT GGG ATC CCC GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGAStop codon Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr AspATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGAStop codons Leu Val Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg AspCTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGAStop codons Leu Val Pro Arg Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr AspCTG GTT CCG CGT GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGAStop codons ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGA CTG ACT GACIle Glu Gly Arg Gly Ile Pro Gly Asn Ser SerStop codons Leu Val Pro Arg Gly Ser Arg Arg Ala Ser ValKinaseCTG GTT CCG CGT GGA TCT CGT CGT GCA TCT GTT GGA TCC CCG GGA ATT CAT CGT GAC TGAStop codons Thrombin↓Thrombin↓Thrombin↓Thrombin↓Factor Xa↓Factor Xa↓Factor Xa↓Factor Xa↓EcoR IBamH I Sma I EcoR IBamH I Sma I pGEX-2T (27-4801-01)CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACGLeu Val Pro Arg Gly Ser Pro Gly Ile His Arg AspStop codons EcoR IThrombin↓BamH I Sma I。
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( )pPICZalphaApGAPZαAPYES2.0pBI121pEGFP-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。
pGEX-TagRFP使用说明
编号 北京华越洋 VECT90032 载体名称 pGEX-TagRFP
pGEX-TagRFP 启动子: 复制子: 质粒分类: 质粒标签: 原核抗性: 克隆菌株: 培养条件: 表达宿主: 诱导方式: 5'测序引物: 3'测序引物: 备注:
pGEX 系列表达载体基本信息： Tac/lac ColE1 ori 大肠杆菌载体；pGEX 系列表达质粒 N-GST，N-TagRFP 氨苄青霉素 Amp DH5α 37℃，有氧，LB 大肠杆菌 IPTG 或乳糖及其类似物 pGEX5: GGGCTGGCAAGCCACGTTTGGTG pGEX3: CCGGGAGCTGCATGTGTCAGAGG 可用 GST 亲和柱纯化重组蛋白
pGEX-20T pGEX-His pGEX-Zsgreen1 pGEX-TagRFP pGEX-6P-3
pGEX-TagRFP 载体质粒图谱和多克隆位点信息 pGEX-TagRFP 载体简介 pGEX-TagRFP 载体序列 pGEX-TagRFP 其他相关 pGEX 系列表达载体：
pGEX-KG pGEX-4T-3 pGEX-4TpGEX-2T pGEX-1λT EcoRI/BAP pGEX-5X-1 pGEX-5X-2 pGEX-5X-3 pGEX-6P-1 pGEX-6P-2
pGEX-4T-1-USP原核表达载体构建
1.
Forward: EcoR I
Reverse: Xho I
2.原核载体的构建
片段的克隆：
载体0.5 2XP pfu buffer 25
Dntps （2.5mM ） 8 Primer-f 1 Primer-R 1 KOD E
1 酶切： dd water 24 10X buffer 4 DNA 10 EcoR I 1
dd water 32.5 10X buffer 4 DNA 2 ug EcoR I 1 Xho I 1 Total
40 ul
37℃，2 h 。
做两个单酶切位点，加上一个未酶切的载体做对照，一起跑胶。
回收。
连接： Vector DNA 2 Insert DNA 6 10X buffer 1.5 T4 DNA ligase
1 For 22℃，
2 h
取7.5 ul 转化涂板。
同时取空载和感受态涂板做对照。
小提鉴定。
测序。
{
⑴冰上20分钟-→热激：42℃、90秒-→冰上2
分钟 ⑵图板（相应抗性的LB 固体平板）
⑶12-16h 后收板，挑单克隆，小提摇菌。
图为：取500 ul菌液，离心加入1Xcracking buffer，13000rpm，离心15min，然后取15ul上样检测，得到如上图，其中L11，L22和L32分子量明显比其他的大，猜测为连接成功的质粒。
然后小提，酶切检测，片段大小为1700bp左右。
确认载体片段插入成功（第四道为空载）。
然后取10ul质粒送测序。
载体图谱
一、pGADT7(Amp)酵母表达载体(AD-Amp)AD forward primer: 5'-TAATACGACTCACTATAGGGC-3'AD reverse primer: 5'-AGATGGTGCACGATGCACAG-3'F：GC AT CGA TAC ATG GC T TG T GCTACTCTGAR：GGCC GGATCC TTAAGAGAGGTAGCTGGGAGAD forward primer: 5'-TAATACGACTCACTATAGGGC-3'AD reverse primer: 5'-AGATGGTGCACGATGCACAG-3'二、pGBKT7(Kan0酵母表达载体(BD-Kan)三、BIFC(Kan)验证互做蛋白表达载体（德国）四、pGR107(Kan)表达载体（PVX-10kb）吴建国MCS:ClaI/SmaI/SalI(pgR107)MCS:ClaI/AscI/NotI/SalI(pgR106) 五、BIFC(Amp)载体（美国）3075（nEYFP-329bp）3076 (cEYFP-218bp)F：GGC GAATTC ATG GCTTGTGCTACTCTGA R：GGC CCTAGG AGAGAGGTAGCTGGGAG TAA TAG TGA六、RNAI(Kan)载体-pFGC5941（王宗华）七、pGEX-4T-1(Amp)原核表达载体八：pET-29a(Kan)原核表达载体九、. pEGAD(Kan)植物表达载体*XbaⅠ cleavage blocked by overlapping dam methylation. Must grow in dam-strain to cut. Note, XhoⅠ is not unique.pTRV2(a) Genome organization of TRV. The TRV-RNA1 open reading frames (ORFs) correspond to 134 and 194 kDa replicase, movement protein (MP) and a 16-kDa cysteine-rich protein. The TRV-RNA2 ORFs corresponds to coat protein (CP), and the 29.4 and 32.8 kDa proteins. gRNA, genomic RNA; sgRNA, subgenomic RNA. Asterisks (*) indicate the readthrough of 134 kDa protein.(b) TRV based VIGS vectors. TRV cDNA clones were placed in betweenthe duplicated CaMV 35S promoter (2×35S) and the nopaline synthase terminator (NOSt) in a T-DNA vector. LB and RB refer to left and right borders of T-DNA. Rz, self-cleaving ribozyme. MCS, multiple cloning sites.十、十一、1103-YFP十二：pBINPLUS十三、pBin438。
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-BpVAX1PBR322pbv220pBluescriptIIKSL4440pCAMBIA-1301pGD926三.PET系列表达载体ProteinExpressionProkaryoticExpressionpETDsbFusionSystems39band40b ProteinExpressionProkaryoticExpressionpETExpressionSystem33b ProteinExpressionProkaryoticExpressionpETExpressionSystems ProteinExpressionProkaryoticExpressionpETExpressionSystemsplusCompetentCells ProteinExpressionProkaryoticExpressionpETGSTFusionSystems41and42 ProteinExpressionProkaryoticExpression ProteinExpressionProkaryoticExpressionpETVectorDNA ProteinPurificationPurificationSystemsStrepTactinResinsandPurificationKits四.PGEX系列表达载体TEcoR pGEX-1I/BAPpGEX-2TpGEX-2TKpGEX-3XpGEX-4T-1pGEX-4T-2pGEX-4T-3pGEX-5X-1pGEX-5X-3 pGEX-6P-1 pGEX-6P-2 pGEX-6P-3五.PTYBsystem PTYB1PTYB2PTYB11PTYB12六.真核表达载体-pPICZalphaA pGAPZαApBI121pEGFP-N1 pEGFP-C1pPIC9K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;一、一个合格质粒的组成要素复制起始位点Ori 即控制复制起始的位点;原核生物DNA分子中只有一个复制起始点;而真核生物DNA分子有多个复制起始位点;抗生素抗性基因可以便于加以检测,如Amp ,Kan多克隆位点MCS克隆携带外源基因片段P/E 启动子/增强子Terms终止信号加polyA信号可以起到稳定mRNA作用二、如何阅读质粒图谱第一步：首先看Ori的位置,了解质粒的类型原核/真核/穿梭质粒第二步：再看筛选标记,如抗性,决定使用什么筛选标记;1Ampr水解β－内酰胺环,解除氨苄的毒性;2tetr可以阻止四环素进入细胞;3camr生成氯霉素羟乙酰基衍生物,使之失去毒性;4neorkanr氨基糖苷磷酸转移酶使G418长那霉素衍生物失活5hygr使潮霉素β失活;第三步：看多克隆位点MCS;它具有多个限制酶的单一切点;便于外源基因的插入;如果在这些位点外有外源基因的插入,会导致某种标志基因的失活,而便于筛选;决定能不能放目的基因以及如何放置目的基因;第四步：再看外源DNA插入片段大小;质粒一般只能容纳小于10Kb的外源DNA片段;一般来说,外源DNA片段越长,越难插入,越不稳定,转化效率越低;第五步：是否含有表达系统元件,即启动子－核糖体结合位点－克隆位点－转录终止信号;这是用来区别克隆载体与表达载体;克隆载体中加入一些与表达调控有关的元件即成为表达载体;选用那种载体,还是要以实验目的为准绳;启动子－核糖体结合位点－克隆位点－转录终止信号启动子－促进DNA转录的DNA顺序,这个DNA区域常在基因或操纵子编码顺序的上游,是DNA 分子上可以与RNApol特异性结合并使之开始转录的部位,但启动子本身不被转录;增强子/沉默子－为真核基因组包括真核病毒基因组中的一种具有增强邻近基因转录过程的调控顺序;其作用与增强子所在的位置或方向无关;即在所调控基因上游或下游均可发挥作用;/沉默子－负增强子,负调控序列;核糖体结合位点/起始密码/SD序列Rbs/AGU/SDs：mRNA有核糖体的两个结合位点,对于原核而言是AUG起始密码和SD序列;转录终止顺序终止子/翻译终止密码子：结构基因的最后一个外显子中有一个AATAAA的保守序列,此位点down－stream有一段GT或T富丰区,这2部分共同构成polyA加尾信号;结构基因的最后一个外显子中有一个AATAAA的保守序列,此位点down－stream有一段GT或T 富丰区,这2部分共同构成polyA加尾信号;回答有人之前提出的一个问题：为什么质粒图谱上有的箭头顺时针有的箭头逆时针,那其实是代表两条DNA链,即质粒是环状双链DNA,它的启动子等在其中一条链上,而它的抗性基因在另一条链上.三、介绍一下关于载体的知识虽然课本上都有写1. 什么是载体即要把一个有用的基因目的基因——研究或应用基因通过基因工程手段送到生物细胞受体细胞,需要运载工具交通工具携带外源基因进入受体细胞,这种运载工具就叫做载体vector;.基因工程所用的vector实际上是DNA分子,是用来携带目的基因片段进入受体细胞的DNA2. 载体的分类―――按功能分成：1克隆载体都有一个松弛的复制子,能带动外源基因,在宿主细胞中复制扩增;它是用来克隆和扩增DNA片段基因的载体;所以有时实验时扩增效率低下,要注意是不是使用的严谨行载体2表达载体具有克隆载体的基本元件ori,Ampr,Mcs等还具有转录/翻译所必需的DNA顺序的载体;―――按进入受体细胞类型分：1原核载体2真核载体3穿梭载体sbuttlevector指在两种宿主生物体内复制的载体分子,因而可以运载目的基因穿梭往返两种生物之间..穿梭质粒含原核和真核生物2个复制子,以确保两类细胞中都能扩增;3. 基因工程载体的3个特点：一都能独立自主的复制：载体DNA分子中有一段不影响它们扩增的非必需区域,如MCS,插在其中的外源DNA片段,能被动的跟着载体一起复制/扩增,就像载体的正常成分一样;二都能便利的加以检测：如载体的药物抗性基因,多是抗生素抗性基因,将受体细胞放在含有该抗生素培养板上培养生长时,只有携带这些抗性基因的载体分子的受体细胞才能存活;三都能容易进入宿主细胞中去,也易从宿主细胞中分离纯化出来;4. 载体的选择和制备：选择载体主要依据构建的目的,同时要考虑载体中应有合适的限制酶切位点;如果构建的目的是要表达一个特定的基因,则要选择合适的表达载体;载体选择主要考虑下述3点：1构建DNA重组体的目的,克隆扩增/表达表达,选择合适的克隆载体/表达载体;2.载体的类型：1克隆载体的克隆能力－据克隆片段大小大选大,小选小;如<10kb选质粒;2表达载体据受体细胞类型－原核/真核/穿梭,哺乳类细胞表达载体;3对原核表达载体应该注意3点：①选择合适的启动子及相应的受体菌；②用于表达真核蛋白质时注意克服4个困难和阅读框错位；③表达天然蛋白质或融合蛋白作为相应载体的参考;3载体MCS中的酶切位点数与组成方向因载体不同而异,适应目的基因与载体易于链接,不产生阅读框架错位;选用质粒最常用做载体的4点要求：①选分子量小的质粒,即小载体1－→不易损坏,在细菌里面拷贝数也多也有大载体；②一般使用松弛型质粒在细菌里扩增不受约束,一般10个以上的拷贝,而严谨型质粒<10个;③必需具备一个以上的酶切位点,有选择的余地；④必需有易检测的标记,多是抗生素的抗性基因,不特指多位Ampr试一试;无论选用哪种载体,首先都要获得载体分子,然后采用适当的限制酶将载体DNA进行切割,获得分子,以便于与目的基因片段进行连接。
质粒载体图谱大全Vector
EMBL-AG
expression vector
pBADM-40(+)
map
araBAD
Amp
N-MBP
TEV
Top10
LMG194
pUC
EMBL-AG
expression vector
pBADM-41(+)
map
araBAD
Amp
N-His
N-MBP
TEV
Top10
LMG194
pUC
EMBL-AG
Cam
C-His
TEV
BL21 (DE3)
p15A
EMBL-AG
expression vector
pACYC-31
map
T7/lac
Cam
N-His
N-GST
TEV
BL21 (DE3)
p15A
EMBL-AG
expression vector
pASK75
tet
Amp
Strep
BL21
Biometra
expression vector
pBAD/His A
map
araBAD
Amp
N-His
EK
Top10
LMG194
pUC
Invitrogen
expression vector
pBAD/His B
map
araBAD
Amp
N-His
EK
Top10
LMG194
pUC
Invitrogen
expression vector
pBAD/His C
pHAT2
map
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
Rosetta系列的表达菌株可以提供T7 RNA聚合酶，它能表达PET系列载体上的外源基因。
pGEX系列载体上的外源基因不需要T7 RNA聚合酶，普通的大肠杆菌经IPTG诱导即可表达Tac启动子是一组由Lac和trp启动子人工构建的杂合启动子，受Lac阻遏蛋白的负调节，它的启动能力比Lac和trp都强。
其中Tac 1是由Trp启动子的－35区加上一个合成的46 bp DNA片段(包括Pribnow 盒)和Lac操纵基因构成，Tac 12是由Trp的启动子-35区和Lac启动子的-10区，加上Lac操纵子中的操纵基因部分和SD序列融合而成蛋白标签：A myc tag is a polypeptide protein tag derived from the c-myc gene product that can be added to a protein using recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits.A myc tag can be used in many different assays that require recognition by an antibody. If there is no antibody against the studied protein, adding a myc-tag allows one to follow the protein with an antibody against the Myc epitope. Examples are cellular localization studies by immunofluorescence or detection by Western blotting.The peptide sequence of the myc-tag is (in 1- and 3-letter codes, respectively): N-EQKLISEEDL-C, N-Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-C, where N stands for Amino-terminus and C stands for Carboxy terminus. The tag is approximately 1202 Daltons in atomic mass and has 10 amino acids.It can be fused to the C-terminus and the N-terminus of a protein. It is advisable not to fuse the tag directly behind the signal peptide of a secretory protein, since it can interfere with translocation into the secretory pathway.A monoclonal antibody against the myc epitope, named 9E10, is available from thenon-commercial Developmental Studies Hybridoma BankpGEX4T1载体基本信息出品公司:GE别名:pGEX-4T-1, pGEX4T1, pGEX 4T 1质粒类型:大肠杆菌蛋白表达载体表达水平:高拷贝启动子:Tac克隆方法:多克隆位点，限制性内切酶载体大小:4969 bp5' 测序引物及序列:pGEX5': GGGCTGGCAAGCCACGTTTGGTG 3' 测序引物及序列:pGEX3': CCGGGAGCTGCATGTGTCAGAGG 载体标签:N-GST载体抗性:Ampicillin 氨苄备注:复制子是pMB1产品目录号:27-4580-01稳定性:瞬时表达Transient组成型:诱导表达病毒/非病毒:非病毒pGEX4T1载体质粒图谱和多克隆位点信息原核生物DNA复制起始点，是DNA链上独特的具有起始DNA复制功能的碱基序列。
大肠杆菌的复制起点包括OriC和OriHThrombin 凝血酶pGEX4T1载体简介pGEX4T1载体序列LOCUS pGEX-4T-1 4969 bp DNA circular SYNDEFINITION pGEX-4T-1ACCESSIONKEYWORDSSOURCEORGANISM other sequences; artificial sequences; vectors.COMMENT This file is created by Vector NTI/COMMENT VNTAUTHORNAME|| FEATURES Location/Qualifierssource 1..4969/organism="pGEX-4T-1"/mol_type="other DNA"promoter 184..212/label="tac_promoter"misc_feature 224..246/label="M13_pUC_rev_primer"gene 258..977/label="GST (variant)"/gene="GST (variant)"CDS 258..977/label="ORF frame 3"misc_feature complement(1019..1041)/label="pGEX_3_primer"promoter 1307..1335/label="AmpR_promoter"gene 1377..2237/label="Ampicillin"/gene="Ampicillin"CDS 1377..2237/label="ORF frame 3"rep_origin 2392..3011/label="pBR322_origin"misc_feature 3309..4400/label="lacI"CDS 3441..4400/label="ORF frame 3"promoter 4449..4478/label="lac_promoter"misc_feature 4492..4514/label="M13_pUC_rev_primer"promoter 4513..4531/label="M13_reverse_primer"CDS 4523..81/label="ORF frame 2"misc_feature 4540..4695/label="lacZ_a"promoter complement(4543..4559)/label="M13_forward20_primer"misc_feature complement(4552..4574)/label="M13_pUC_fwd_primer"pGEX4T2载体基本信息出品公司:GE别名:pGEX-4T-2, pGEX4T2, pGEX 4T 2质粒类型:大肠杆菌蛋白表达载体表达水平:高拷贝启动子:Tac克隆方法:多克隆位点，限制性内切酶载体大小:4970 bp5' 测序引物及序列:pGEX5': GGGCTGGCAAGCCACGTTTGGTG 3' 测序引物及序列:pGEX3': CCGGGAGCTGCATGTGTCAGAGG 载体标签:N-GST载体抗性:Ampicillin 氨苄备注:复制子是pMB1产品目录号:27-4581-01稳定性:瞬时表达Transient组成型:诱导表达病毒/非病毒:非病毒pGEX4T2载体质粒图谱和多克隆位点信息pGEX4T2载体简介pGEX4T2载体序列LOCUS pGEX-4T-2 4970 bp DNA circular SYN DEFINITION pGEX-4T-2ACCESSIONKEYWORDSSOURCEORGANISM other sequences; artificial sequences; vectors.COMMENT This file is created by Vector NTI/COMMENT VNTAUTHORNAME||FEATURES Location/Qualifierssource 1..4970/organism="pGEX-4T-2"/mol_type="other DNA"promoter 184..212/label="tac_promoter"misc_feature 224..246/label="M13_pUC_rev_primer"gene 258..978/label="GST (variant)"/gene="GST (variant)"CDS 258..974/label="ORF frame 3"misc_feature complement(1020..1042)/label="pGEX_3_primer"promoter 1308..1336/label="AmpR_promoter"gene 1378..2238/label="Ampicillin"/gene="Ampicillin"CDS 1378..2238/label="ORF frame 1"rep_origin 2393..3012/label="pBR322_origin"misc_feature 3310..4401/label="lacI"CDS 3442..4401/label="ORF frame 1"promoter 4450..4479/label="lac_promoter"misc_feature 4493..4515/label="M13_pUC_rev_primer"promoter 4514..4532/label="M13_reverse_primer"CDS 4524..81/label="ORF frame 3"misc_feature 4541..4696/label="lacZ_a"promoter complement(4544..4560)/label="M13_forward20_primer"misc_feature complement(4553..4575)/label="M13_pUC_fwd_primer" pGEX4T3载体质粒图谱和多克隆位点信息pGEX4T3载体简介pGEX4T3载体序列LOCUS pGEX-4T-3 4968 bp DNA circular SYN DEFINITION pGEX-4T-3ACCESSIONKEYWORDSSOURCEORGANISM other sequences; artificial sequences; vectors.COMMENT This file is created by Vector NTI/COMMENT VNTAUTHORNAME||FEATURES Location/Qualifierssource 1..4968/organism="pGEX-4T-3"/mol_type="other DNA" promoter 184..212/label="tac_promoter"misc_feature 224..246/label="M13_pUC_rev_primer" gene 258..976/label="GST (variant)"/gene="GST (variant)"CDS 258..980/label="ORF frame 3"misc_feature complement(1018..1040)/label="pGEX_3_primer" promoter 1306..1334/label="AmpR_promoter" gene 1376..2236/label="Ampicillin"/gene="Ampicillin"CDS 1376..2236/label="ORF frame 2"rep_origin 2391..3010/label="pBR322_origin"misc_feature 3308..4399/label="lacI"CDS 3440..4399/label="ORF frame 2"promoter 4448..4477/label="lac_promoter"misc_feature 4491..4513/label="M13_pUC_rev_primer"promoter 4512..4530/label="M13_reverse_primer"CDS 4522..81/label="ORF frame 1"misc_feature 4539..4694/label="lacZ_a"promoter complement(4542..4558)/label="M13_forward20_primer"misc_feature complement(4551..4573)/label="M13_pUC_fwd_primer" pGEX6P1载体基本信息出品公司:GE别名:pGEX-6P-1, pGEX6P1, pGEX 6P 1质粒类型:大肠杆菌蛋白表达载体表达水平:高拷贝启动子:Tac克隆方法:多克隆位点，限制性内切酶载体大小:4984 bp5' 测序引物及序列:pGEX5': GGGCTGGCAAGCCACGTTTGGTG 3' 测序引物及序列:pGEX3': CCGGGAGCTGCATGTGTCAGAGG 载体标签:N-GST载体抗性:Ampicillin 氨苄备注:复制子是pMB1产品目录号:27-4597-01稳定性:瞬时表达Transient组成型:诱导表达病毒/非病毒:非病毒pGEX6P1载体质粒图谱和多克隆位点信息pGEX6P1载体简介pGEX6P1载体序列LOCUS pGEX-6P-1 4984 bp DNA circular SYN DEFINITION pGEX-6P-1ACCESSIONKEYWORDSSOURCEORGANISM other sequences; artificial sequences; vectors.COMMENT This file is created by Vector NTI/COMMENT VNTAUTHORNAME||FEATURES Location/Qualifierssource 1..4984/organism="pGEX-6P-1"/mol_type="other DNA" promoter 184..212/label="tac_promoter"misc_feature 224..246/label="M13_pUC_rev_primer" gene 258..992/label="GST"/gene="GST"CDS 258..992/label="ORF frame 3"misc_feature 918..938/label="precision"misc_feature complement(1034..1056)/label="pGEX_3_primer" promoter 1322..1350/label="AmpR_promoter" gene 1392..2252/label="Ampicillin"/gene="Ampicillin"CDS 1392..2252/label="ORF frame 3"rep_origin 2407..3026/label="pBR322_origin"misc_feature 3324..4415/label="lacI"CDS 3456..4415/label="ORF frame 3"promoter 4464..4493/label="lac_promoter"misc_feature 4507..4529/label="M13_pUC_rev_primer"promoter 4528..4546/label="M13_reverse_primer"CDS 4538..81/label="ORF frame 2"misc_feature 4555..4710/label="lacZ_a"promoter complement(4558..4574)/label="M13_forward20_primer"misc_feature complement(4567..4589)/label="M13_pUC_fwd_primer"pET28a载体基本信息出品公司:EMD Biosciences (Novagen)别名:pET28a, pet 28a, pET-28a(+)质粒类型:大肠杆菌蛋白表达表达水平:高克隆方法:多克隆位点，限制性内切酶载体大小:5369 bp5' 测序引物及序列:T7: 5'-TAATACGACTCACTATAGGG-3'3' 测序引物序列: T7t: 5'-GCTAGTTATTGCTCAGCGG-3'载体标签:N-His, N-Thrombin, N-T7, C-His载体抗性:卡那N端含有Thrombin蛋白酶切位点; pET28a, b, c的差备注:异仅仅存在于多克隆位点处。