Human CDKL5 ORF clone (NM_001037343) (Cat. No.: V022401)

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Basic Information

Name:: pAAV-CDKL5(human)-mCherry-FLAG

Accession ID:: NM_001037343.2

Antibiotic Resistance:: Ampicillin

Length:: 8514 bp

Type:: Protein expression

Replication origin:: ori

Host:: Mammalian cells, Adeno-associated virus

Selection Marker:: mCherry

Promoter:: CMV

Fusion Tag:: mCherry-FLAG

Expression Method:: Transient

Gene Synonyms:: CFAP247; DEE2; EIEE2; ISSX; STK9

Transcript Definition:: Homo sapiens cyclin dependent kinase like 5 (CDKL5), transcript variant II, mRNA

Cat. No.: V022401 pAAV-CDKL5(human)-mCherry-FLAG

$ 298.4

In stock, 1-2 weeks for quality controls

Two tubes of transfection grade plasmid, each tube is about 100 µg (lyophilized).

Resources

Transcript ID

NM_001037343.2 NM_003159.3

Fusion Tag

mCherry-FLAG HA

Selection Marker

mCherry Neo/G418

Expression Method

Transient

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.

Human CDKL5 ORF clone (NM_001037343) (Cat. No.: V022401) Sequence

LOCUS       V022401                 8514 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V022401
VERSION     V022401
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"
     CDS             1338..2045
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /transl_table=1
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
                     /label="mCherry"
     CDS             2046..2102
                     /codon_start=1
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /transl_table=1
                     /translation="ATNFSLLKQAGDVEENPGP"
                     /label="P2A"
     CDS             2109..5198
                     /label="CDKL5(NM_001037343)"
                     /note="CDKL5(NM_001037343)"
                     /gene="CDKL5"
     CDS             5199..5222
                     /codon_start=1
                     /product="FLAG® epitope tag, followed by an enterokinase
                     cleavage site"
                     /transl_table=1
                     /translation="DYKDDDDK"
                     /label="FLAG"
     polyA_signal    5261..5737
                     /note="human growth hormone polyadenylation signal"
                     /label="hGH poly(A) signal"
     repeat_region   5777..5917
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /rpt_type=inverted
                     /label="AAV2 ITR"
     rep_origin      5992..6447
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        6729..6833
                     /gene="bla"
                     /label="AmpR promoter"
     CDS             6834..7694
                     /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      7865..8453
                     /direction=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"