3XFlag-pA-Tn5-Fl vector (V004204)

Basic Vector Information

      • Vector Name:
      • 3XFlag-pA-Tn5-Fl
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 8679 bp
      • Type:
      • Bacterial Expression
      • Promoter:
      • T7
      • Cloning Method:
      • Restriction Enzyme
      • 5' Primer:
      • CGG TTT AAA CCG GGG ATC TCG
      • 3' Primer:
      • TTG CGC CGC AAC ATT CAC C

3XFlag-pA-Tn5-Fl vector Vector Map

3XFlag-pA-Tn5-Fl8679 bp4008001200160020002400280032003600400044004800520056006000640068007200760080008400pGEX 3'lacIlacI promoterrrnB T1 terminatorrrnB T1 terminatorrrnB T1 terminatorrrnB T1 terminatorrrnB T1 terminatorT7lac operatorRBS3xFLAGprotein ATn5 transposaseMxe GyrA inteinCBDT7 TermAmpR promoterAmpRM13 orioriL4440pRS-marker

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

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                8679 bp ds-DNA     circular SYN 26-MAY-2021
DEFINITION  Expresses protein A and Tn5 Transposase fusion protein in bacterial 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    3XFlag-pA-Tn5-Fl
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)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2019-04-29"; volume: "10(1):1930. doi"; pages: " 
            10.1038/s41467-019-09982-5"
FEATURES             Location/Qualifiers
     source          1..8679
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     126..148
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(142..333)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(890..1972)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1934..1953
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1973..2050)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      2203..2289
                     /gene="Escherichia coli rrnB"
                     /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      2935..3021
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3156..3175
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3156..3174
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3175..3199
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3249..3314
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3333..3758
                     /codon_start=1
                     /product="immunoglobulin binding domains of Staphylocuccus 
                     aureus protein A"
                     /label=protein A
                     /translation="SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHL
                     PNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP
                     NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK"
     CDS             3840..5267
                     /codon_start=1
                     /product="transposase from the bacterial Tn5 transposon 
                     (Reznikoff, 1993)"
                     /label=Tn5 transposase
                     /translation="MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKS
                     ITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYR
                     HQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESG
                     KWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGL
                     YLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNA
                     VLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAER
                     QRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTP
                     DECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQS
                     KLDGFLAAKDLMAQGIKI"
     CDS             5268..5861
                     /codon_start=1
                     /gene="Mycobacterium xenopi gyrA"
                     /product="modified GyrA intein (Southworth et al., 1999)"
                     /label=Mxe GyrA intein
                     /note="N-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
                     LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
                     IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
                     AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
     CDS             5892..6047
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     primer_bind     complement(6112..6130)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6126..6173
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        6233..6337
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6338..7198
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6556..6575)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(7240..7753)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7435..7456)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7647..7666
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      7864..8452
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8353..8372
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     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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