Basic Vector Information
- Vector Name:
- pET-35b(+)
- Antibiotic Resistance:
- Kanamycin
- Length:
- 5927 bp
- Type:
- E. coli Expression Vectors
- Replication origin:
- ori
- Promoter:
- T7-lac
- Cloning Method:
- Enzyme digestion and ligation
- 5' Primer:
- T7
- 3' Primer:
- 5'-TAATACGACTCACTATAGGG-3'
- Fusion Tag:
- N-CBD|clos|,C-His
pET-35b(+) 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 ℃.
pET-35b(+) vector Sequence
LOCUS 40924_17916 5927 bp DNA circular SYN 13-JAN-2022 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5927) TITLE Direct Submission REFERENCE 2 (bases 1 to 5927) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..5927 /mol_type="other DNA" /organism="synthetic DNA construct" terminator complement(26..73) /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(150..173) /codon_start=1 /label=8xHis /note="8xHis affinity tag" /translation="HHHHHHHH" CDS complement(249..260) /codon_start=1 /label=Factor Xa site /note="Factor Xa recognition and cleavage site" /translation="IEGR" CDS complement(291..335) /codon_start=1 /label=S-Tag /note="affinity and epitope tag derived from pancreatic ribonuclease A" /translation="KETAAAKFERQHMDS" CDS complement(351..368) /codon_start=1 /label=thrombin site /note="thrombin recognition and cleavage site" /translation="LVPRGS" RBS complement(860..882) /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind complement(897..921) /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(922..940) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 1253..1330 /label=lacI promoter CDS 1331..2410 /codon_start=1 /label=lacI /note="lac repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" protein_bind 2426..2447 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS 3222..3410 /codon_start=1 /label=rop /note="Rop protein, which maintains plasmids at low copy number" /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" misc_feature 3515..3657 /label=bom /note="basis of mobility region from pBR322" rep_origin complement(3843..4431) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS 4553..5365 /codon_start=1 /label=KanR /note="aminoglycoside phosphotransferase" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" rep_origin complement(5461..5916) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
This page is informational only.