pAAV-EF1a-S-HBs vector (Cat. No.: V046733)

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Basic Information

Name:: pAAV-EF1a-S-HBs

Length:: 6566 bp

Type:: Protein expression

Replication origin:: ori

Host:: Mammalian cells, Adeno-associated virus

Copy Number:: High

Promoter:: EF1a

5' Primer:: EF-1α promoter-F3

Growth Temperature:: 37℃

Expression Method:: Transient

$ 198.7

In stock, 1 week for quality controls

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

pAAV-EF1a-S-HBs vector (Cat. No.: V046733) Sequence

LOCUS       pAAV-EF1a-S-HBs         6566 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V046733
VERSION     V046733
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     repeat_region   1..141
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /rpt_type=inverted
                     /label="AAV2 ITR"
     enhancer        221..524
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        525..727
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     intron          876..1254
                     /note="fusion between human cytomegalovirus intron A and
                     human β-globin intron 2"
                     /label="chimeric intron"
     promoter        1332..2510
                     /note="strong constitutive promoter for human elongation
                     factor EF-1α"
                     /label="EF-1α promoter"
     CDS             2572..3252
                     /codon_start=1
                     /note="hepatitis B surface antigen"
                     /product="major/small (S) envelope protein of hepatitis B
                     virus"
                     /transl_table=1
                     /translation="MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGT
                     TVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGML
                     PVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLW
                     EWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCL
                     WVYI*"
                     /label="HBsAg (S)"
     polyA_signal    3313..3789
                     /note="human growth hormone polyadenylation signal"
                     /label="hGH poly(A) signal"
     repeat_region   3829..3969
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /rpt_type=inverted
                     /label="AAV2 ITR"
     rep_origin      4044..4499
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        4781..4885
                     /gene="bla"
                     /label="AmpR promoter"
     CDS             4886..5746
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     rep_origin      5917..6505
                     /direction=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"