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pAAV-TBG-MCS-shRNA-ZsGreen1 vector (V015050) Gene synthesis in pAAV-TBG-MCS-shRNA-ZsGreen1 backbone

Price Information

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V015050 pAAV-TBG-MCS-shRNA-ZsGreen1 In stock, instant shipping

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

Basic Vector Information

pAAV - TBG - MCS - shRNA - ZsGreen1 is an adeno-associated virus-based plasmid for gene silencing by shRNA in the presence of the TBG promoter, and ZsGreen1 is used as a reporter gene to monitor the progress of the experiment.

Vector Name:
pAAV-TBG-MCS-shRNA-ZsGreen1
Antibiotic Resistance:
Ampicillin
Length:
5847 bp
Type:
RNA interference
Replication origin:
ori
Host:
Mammalian cells, Adeno-associated virus
Promoter:
TBG
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

pAAV-TBG-MCS-shRNA-ZsGreen1 vector Map

Copy Sequence View Map
pAAV-TBG-MCS-shRNA-ZsGreen15847 bp60012001800240030003600420048005400AAV2 ITRCMV enhancerCMV promoterchimeric intronZsGreen1hGH poly(A) signalTBG promoterAAV2 ITRf1 oriAmpR promoterAmpRori

Plasmid 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 ℃.

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-TBG-MCS-shRNA-ZsGreen1 vector Sequence

LOCUS       62056_2515        5847 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5847)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5847
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     enhancer        221..524
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        525..728
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     intron          877..1255
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and
                     human beta-globin intron 2"
     CDS             1337..2029
                     /codon_start=1
                     /label=ZsGreen1
                     /note="Zoanthus green fluorescent protein"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     polyA_signal    2041..2517
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     promoter        2628..3037
                     /label=TBG promoter
                     /note="promoter from the human thyroxine-binding globulin
                     gene (Yan et al., 2012)"
     repeat_region   3110..3250
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      3325..3780
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4062..4166
                     /label=AmpR promoter
     CDS             4167..5024
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5198..5786
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"