Basic Vector Information
3XFlag-pA-Tn5-Fl 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 ℃.
3XFlag-pA-Tn5-Fl vector Sequence
LOCUS 40924_80 8679 bp DNA circular SYN 26-MAY-2021 DEFINITION Expresses protein A and Tn5 Transposase fusion protein in bacterial cells. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8679) AUTHORS Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG, Ahmad K, Henikoff S TITLE CUT JOURNAL Nat Commun. 2019 Apr 29;10(1):1930. doi: 10.1038/s41467-019-09982-5. PUBMED 31036827 REFERENCE 2 (bases 1 to 8679) TITLE Direct Submission REFERENCE 3 (bases 1 to 8679) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat Commun."; date: "2019-04-29"; pages: " 10.1038/s41467-019-09982-5" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..8679 /mol_type="other DNA" /organism="synthetic DNA construct" primer_bind 126..148 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(145..333) /codon_start=1 /label=rop /note="Rop protein, which maintains plasmids at low copy number" /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" protein_bind complement(856..877) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS complement(893..1972) /codon_start=1 /label=lacI /note="lac repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" promoter complement(1973..2050) /label=lacI promoter terminator 2203..2289 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 2386..2472 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 2569..2655 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 2752..2838 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator complement(2938..2981) /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" promoter 3156..3174 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 3175..3199 /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)." RBS 3214..3236 /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 3249..3314 /codon_start=1 /label=3xFLAG /note="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 3333..3758 /codon_start=1 /label=protein A /note="immunoglobulin binding domains of Staphylocuccus aureus protein A" /translation="SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHL PNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK" CDS 3840..5267 /codon_start=1 /label=Tn5 transposase /note="transposase from the bacterial Tn5 transposon (Reznikoff, 1993)" /translation="MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKS ITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYR HQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESG KWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGL YLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNA VLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAER QRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTP DECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQS KLDGFLAAKDLMAQGIKI" CDS 5268..5861 /codon_start=1 /label=Mxe GyrA intein /note="modified GyrA intein (Southworth et al., 1999)" /translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA" CDS 5892..6047 /codon_start=1 /label=CBD /note="chitin binding domain from chitinase A1 (Watanabe et al., 1994)" /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV PALWQLQ" terminator 6126..6173 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter 6233..6337 /label=AmpR promoter CDS 6338..7195 /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin complement(7240..7753) /direction=LEFT /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" rep_origin 7864..8452 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8606..8623 /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(8645..8664) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker"
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