pMRE105_PnptII_eYFP 载体 (V004476)

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。

载体名称:
pMRE105_PnptII_eYFP
载体抗性:
Ampicillin
载体长度:
13484 bp
载体类型:
Tn7 delivery vector
复制子:
pSC101 ori
载体来源:
Remus-Emsermann MNP., Gisler P, Drissner D.
启动子:
araBAD

pMRE105_PnptII_eYFP 载体图谱

pMRE105_PnptII_eYFP13484 bp600120018002400300036004200480054006000660072007800840090009600102001080011400120001260013200araCaraBAD promoterTransposon Tn7 transposition protein TnsBTnsDrrnB T1 terminatorrrnB T2 terminatorAmpR promoterAmpRoriTpSC101 oriRep101(Ts)Tn7LEYFPTn7 right end

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。

pMRE105_PnptII_eYFP 载体序列

LOCUS       V004476                13484 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004476
VERSION     V004476
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 13484)
  AUTHORS   Remus-Emsermann MNP., Gisler P, Drissner D.
  TITLE     MiniTn7-transposon delivery vectors for inducible or constitutive
            fluorescent protein expression in Enterobacteriaceae
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13484)
  AUTHORS   Remus-Emsermann MNP., Drissner D.
  TITLE     Direct Submission
  JOURNAL   Submitted (23-MAR-2016) Institute for Food Sciences, Agroscope,
            Schloss 1, Waedenswil, Zurich 8820, Switzerland
REFERENCE   3  (bases 1 to 13484)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 13484)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName:
            "Unpublished"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (23-MAR-2016) Institute for Food Sciences, Agroscope, Schloss 1,
            Waedenswil, Zurich 8820, Switzerland"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..13484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(149..1024)
                     /label="araC"
                     /note="L-arabinose regulatory protein"
     promoter        1051..1335
                     /label="araBAD promoter"
                     /note="promoter of the L-arabinose operon of E. coli; the
                     araC regulatory gene is transcribed in the opposite
                     direction (Guzman et al., 1995)"
     CDS             1453..2271
                     /gene="tnsA"
                     /label="Transposon Tn7 transposition protein TnsA"
                     /note="Transposon Tn7 transposition protein TnsA from
                     Escherichia coli. Accession#: P13988"
     CDS             2261..4366
                     /gene="tnsB"
                     /label="Transposon Tn7 transposition protein TnsB"
                     /note="Transposon Tn7 transposition protein TnsB from
                     Escherichia coli. Accession#: P13989"
     CDS             4366..6030
                     /gene="tnsC"
                     /label="Transposon Tn7 transposition protein TnsC"
                     /note="Transposon Tn7 transposition protein TnsC from
                     Escherichia coli. Accession#: P05846"
     CDS             6036..7562
                     /codon_start=1
                     /gene="tnsD"
                     /product="TnsD"
                     /label="tnsD"
                     /note="Tn7 transposition site-selector"
                     /protein_id="AMZ00009.1"
                     /translation="MRNFPVPYSNELIYSTIARAGVYQGIVSPKQLLDEVYGNRKVVAT
                     LGLPSHLGVIARHLHQTGRYAVQQLIYEHTLFPLYAPFVGKERRDEAIRLMEYQAQGAV
                     HLMLGVAASRVKSDNRFRYCPDCVALQLNRYGEAFWQRDWYLPALPYCPKHGALVFFDR
                     AVDDHRHQFWALGHTELLSDYPKDSLSQLTALAAYIAPLLDAPRAQELSPSLEQWTLFY
                     QRLAQDLGLTKSKHIRHDLVAERVRQTFSDEALEKLDLKLAENKDTCWLKSIFRKHRKA
                     FSYLQHSIVWQALLPKLTVIEALQQASALTEHSITTRPVSQSVQPNSEDLSVKHKDWQQ
                     LVHKYQGIKAARQSLEGGVLYAWLYRHDRDWLVHWNQQHQQERLAPAPRVDWNQRDRIA
                     VRQLLRIIKRLDSSLDHPRATSSWLLKQTPNGTSLAKNLQKLPLVALCLKRYSESVEDY
                     QIRRISQAFIKLKQEDVELRRWRLLRSATLSKERITEEAQRFLEMVYGEE"
     gene            6036..7562
                     /gene="tnsD"
                     /label="tnsD"
     terminator      8196..8282
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      8374..8401
                     /label="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        8420..8511
                     /label="AmpR promoter"
     CDS             8512..9369
                     /label="AmpR"
                     /note="beta-lactamase"
     oriT            complement(9543..9652)
                     /direction=LEFT
                     /label="oriT"
                     /note="incP origin of transfer"
     rep_origin      10392..10614
                     /label="pSC101 ori"
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             10662..11609
                     /label="Rep101(Ts)"
                     /note="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
     mobile_element  12045..12210
                     /label="Tn7L"
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     CDS             12509..13225
                     /label="EYFP"
                     /note="enhanced YFP"
     repeat_region   13273..13484
                     /label="Tn7 right end"
                     /note="Tn7 right end"