pcDNA-K-GECO1 vector (V000025) Gene synthesis in pcDNA-K-GECO1 backbone

COA reports Sequencing Results MSDS Download GenBank File(.gb)

Price Information

Cat No.	Plasmid Name	Availability	Buy one, get one free! (?)
V000025	pcDNA-K-GECO1	In stock, instant shipping

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

Basic Vector Information

Mammalian cell expression plasmid for red fluorescent Ca2+ indicator K-GECO1

Vector Name:: pcDNA-K-GECO1

Antibiotic Resistance:: Ampicillin

Length:: 6598 bp

Type:: Mammalian Expression

Replication origin:: ori

Copy Number:: High Copy

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

pcDNA-K-GECO1 vector Map

Copy Sequence View Map

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.

References

Shen et al BMC Biol. 2018 Jan 16;16(1):9. doi: 10.1186/s12915-018-0480-0.

A genetically encoded Ca(2+) indicator based on circularly permutated sea anemone red fluorescent protein eqFP578. Shen Y, Dana H, Abdelfattah AS, Patel R, Shea J, Molina RS, Rawal B, Rancic V, Chang YF, Wu L, Chen Y, Qian Y, Wiens MD, Hambleton N, Ballanyi K, Hughes TE, Drobizhev M, Kim DS, Koyama M, Schreiter ER, Campbell RE. BMC Biol. 2018 Jan 16;16(1):9. doi: 10.1186/s12915-018-0480-0. 10.1186/s12915-018-0480-0 PubMed 29338710

pcDNA-K-GECO1 vector Sequence

LOCUS       40924_9846        6598 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Mammalian cell expression plasmid for red fluorescent Ca2+ indicator
            K-GECO1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6598)
  AUTHORS   Shen Y, Dana H, Abdelfattah AS, Patel R, Shea J, Molina RS, Rawal B,
            Rancic V, Chang YF, Wu L, Chen Y, Qian Y, Wiens MD, Hambleton N, 
            Ballanyi K, Hughes TE, Drobizhev M, Kim DS, Koyama M, Schreiter ER, 
            Campbell RE
  TITLE     A genetically encoded Ca(2+) indicator based on circularly 
            permutated sea anemone red fluorescent protein eqFP578.
  JOURNAL   BMC Biol. 2018 Jan 16;16(1):9. doi: 10.1186/s12915-018-0480-0.
  PUBMED    29338710
REFERENCE   2  (bases 1 to 6598)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6598)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC Biol.";
            date: "2018-01-16"; pages: "
            10.1186/s12915-018-0480-0"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        490..693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        738..756
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      781..790
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2070..2294
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2340..2768
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2782..3111
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             3178..3969
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    4146..4279
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4316..4332)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4340..4356)
                     /label=lac operator
                     /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)."
     promoter        complement(4364..4394)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4409..4430)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(4547..4564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4718..5306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5480..6337)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6338..6442)
                     /label=AmpR promoter
     primer_bind     complement(6517..6536)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"