pET32 EKLIC载体序列及图谱文档格式.docx

pET32 EKLIC载体序列及图谱文档格式.docx

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pBRrevBam_primer904885

tet（576-851）23292604

ROP30793270

pGEX_3_primer32863264

pBR322_origin43043685

Ampicillin53194459

AmpR_promoter53895361

ORFStartEnd

ORFframe1709137

ORFframe23441438

ORFframe313112270

ORFframe253194459

EnzymeNameCut

XhoI158

NotI166

EagI166

HindIII173

SalI179

SacI190

EcoRI192

BamHI198

EcoRV206

NcoI211

XmaI228

SmaI230

KpnI255

BglII258

BstBI285

MscI368

XbaI746

BclI1552

ApaI1749

HpaI2044

PstI4777

pET32EK/LIC载体简介

ThepET-32Ek/LICvectorispreparedforrapid,directionalcloningofPCR-amplifiedDNAforhigh-levelexpressionofpolypeptidesfusedwiththe109aaTrx•Tag™thioredoxinprotein.UsingspecificallydesignedprimersforamplificationandthepET-32Ek/LICCloningKit（Cat.No.69076-3）,insertscanbeefficientlyclonedwithouttheneedforrestrictiondigestionorligation.FusionproteinsalsocontaincleavableHis•Tag®

andS•Tag™sequencesfordetectionandpurification.Uniquesitesareshownonthecirclemap.NotethatthesequenceisnumberedbythepBR322convention,sotheT7expressionregionisreversedonthecirclemap.Thecloning/expressionregionofthecodingstrandtranscribedbyT7RNApolymeraseisshownbelow.Thef1originisorientedsothatinfectionwithhelperphagewillproducevirionscontainingsinglestrandedDNAthatcorrespondstothecodingstrand.Therefore,singlestrandedsequencingshouldbeperformedusingtheT7terminatorprimer（Cat.No.69337-3）.

pET32EK/LIC载体序列

LOCUSpET-32Ek/LIC5917bpDNACircularSYN

DEFINITIONpET-32Ek/LIC

ACCESSION

KEYWORDS

SOURCE

ORGANISMothersequences;

artificialsequences;

vectors.

COMMENTThisfileiscreatedbyVectorNTI

COMMENTORIGDB|GenBank

COMMENTVNTAUTHORNAME||

COMMENTVNTOAUTHORAD1|NovagenInc.|

FEATURESLocation/Qualifiers

source1..5917

/organism="

pET-32Ek/LIC"

/mol_type="

otherDNA"

terminatorcomplement（1..129）

/label="

T7_terminator"

misc_feature69..87

T7_Terminal_primer"

CDScomplement（137..709）

ORFframe1"

/translation="

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILD

EIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKE

FLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKSP

GFSSTMAISDPNSSSVDKLAAALEHHHHHH*"

misc_featurecomplement（236..250）

EK"

misc_featurecomplement（266..310）

S15"

misc_feature319..2270

lacI"

CDS344..1438

ORFframe2"

MMMMMVHMARTRTGQVSVEELFQLTFRQCTHFGCRHFTVFEQQQ

SRDTTDAIFRRSARVLIDVQFCNGQFALIFVSDFIQNRGDHFARTAPLCPEIDEDRPV

RFEYIRVKTVVSQVNNFIAHMYISFLKLNKIISRGELLSAHNSPIVSRINFAGSRSIS

ILYAGRIVAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLG

AISLHAPFLAAAVLNGLNLLLGCFLMQESHKGERRDPGHHRMAQNLSRYGMIAPGRES

IQGGECETSNVIRCRRVCRCLLSDRFPRGEPGQPRFCENAGKSGSGDGGAELHSQPRG

TTTGGQTVVADWRCHLQSGPARAVANCRGD*"

misc_featurecomplement（344..361）

6xHIS"

misc_featurecomplement（718..734）

T7_transl_en_RBS"

misc_featurecomplement（752..779）

lacO"

promotercomplement（779..797）

T7_promoter"

gene833..1096

tet（300-563）"

/gene="

misc_featurecomplement（885..904）

pBRrevBam_primer"

CDS1311..2270

ORFframe3"

MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKS

RADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNV

PALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGW

HKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITE

SGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQ

LLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"

gene2329..2604

tet（576-851）"

misc_feature3079..3270

ROP"

misc_featurecomplement（3264..3286）

pGEX_3_primer"

rep_origincomplement（3685..4304）

pBR322_origin"

genecomplement（4459..5319）

Ampicillin"

CDScomplement（4459..5319）

MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY

IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE

YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL

DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL

LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA

EIGASLIKHW*"

promotercomplement（5361..5389）

AmpR_promoter"

ORIGIN

1atccggatatagttcctcctttcagcaaaaaacccctcaagacccgtttagaggccccaa

61ggggttatgctagttattgctcagcggtggcagcagccaactcagcttcctttcgggctt

121tgttagcagccggatctcagtggtggtggt