Basic Vector Information
pTag 2.0 vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
pTag 2.0 vector Sequence
LOCUS 40924_42534 6284 bp DNA circular SYN 18-DEC-2018 DEFINITION Cloning vector pTag 2.0, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6284) AUTHORS Vidigal J, Fernandes B, Dias MM, Patrone M, Roldao A, Carrondo MJT., Alves PM, Teixeira AP. TITLE RMCE-based insect cell platform to produce membrane proteins captured on HIV-1 Gag virus-like particles JOURNAL Appl. Microbiol. Biotechnol. (2017) In press PUBMED 29143881 REFERENCE 2 (bases 1 to 6284) AUTHORS Vidigal J. TITLE Direct Submission JOURNAL Submitted (30-OCT-2017) Animal Cell Technology Unit, Ibet, Av. da Republica, ITQB building, Oeiras 2780-157, Portugal REFERENCE 3 (bases 1 to 6284) TITLE Direct Submission REFERENCE 4 (bases 1 to 6284) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Appl. Microbiol. Biotechnol. (2017) In press" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (30-OCT-2017) Animal Cell Technology Unit, Ibet, Av. da Republica, ITQB building, Oeiras 2780-157, Portugal" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..6284 /mol_type="other DNA" /organism="synthetic DNA construct" primer_bind 379..395 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 443..990 /label=OpIE-2 promoter /note="strong constitutive baculovirus promoter for insect cell expression" protein_bind 1027..1074 /label=FRT /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." CDS 1101..1817 /codon_start=1 /label=EGFP /note="enhanced GFP" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" polyA_signal 1972..2027 /label=beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" polyA_signal 2442..2563 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" CDS complement(2591..3613) /codon_start=1 /label=HygR /note="aminoglycoside phosphotransferase from E. coli" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" promoter complement(3704..3995) /label=OpIE-1 promoter /note="moderate constitutive baculovirus promoter for insect cell expression" primer_bind complement(4063..4079) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(4087..4103) /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)." promoter complement(4111..4141) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(4156..4177) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(4465..5053) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5227..6084) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6085..6189) /label=AmpR promoter
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