Human MCL1 ORF clone (NM_021960) (Cat. No.: V032804)

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Basic Information

Name:: pT3-EF1a-MCL1(human)

Accession ID:: NM_021960.5

Antibiotic Resistance:: Ampicillin

Length:: 6461 bp

Type:: Protein expression

Replication origin:: ori

Host:: Mammalian cells

Promoter:: EF1a

5' Primer:: PEF-F

Growth Strain(s):: DH5a

Growth Temperature:: 37℃

Expression Method:: Transient

Gene Synonyms:: bcl2-L-3; BCL2L3; EAT; Mcl-1; MCL1-ES; mcl1/EAT; MCL1L; MCL1S; TM

Transcript Definition:: Homo sapiens MCL1 apoptosis regulator, BCL2 family member (MCL1), transcript variant 1, mRNA

Cat. No.: V032804 pT3-EF1a-MCL1(human)

$ 298.8

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_021960.5

Fusion Tag

3xFLAG 3xMyc

Selection Marker

Puro Neo/G418

Expression Method

Constiutive, Stable / Transient 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 MCL1 ORF clone (NM_021960) (Cat. No.: V032804) Sequence

LOCUS       pT3-EF1a-MCL1(human)    6461 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V032804
VERSION     V032804
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     promoter        1..1179
                     /note="strong constitutive promoter for human elongation
                     factor EF-1α"
                     /label="EF-1α promoter"
     promoter        1196..1214
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     CDS             1306..2355
                     /label="MCL1(NM_021960)"
                     /note="MCL1(NM_021960)"
                     /gene="MCL1"
     polyA_signal    2389..2613
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     protein_bind    2683..2716
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (ATGTATGC) (Shaw et al., 2021)."
                     /label="loxP"
     primer_bind     complement(3174..3190)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 fwd"
     promoter        3664..3768
                     /gene="bla"
                     /label="AmpR promoter"
     CDS             3769..4629
                     /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      4800..5388
                     /direction=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     protein_bind    5676..5697
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     promoter        5712..5742
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5750..5766
                     /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-β-D-thiogalactopyranoside (IPTG)."
                     /label="lac operator"
     primer_bind     5774..5790
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6288..6321
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (ATGTATGC) (Shaw et al., 2021)."
                     /label="loxP"