Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V008675 | pCC1BAC | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pCC1BAC is a single copy vector that is inducible with 0.04% Arabinose or 1x CopyControl Induction Solution (CopyControl Fosmid clones grown in TransforMax EPI300 cells can be amplified to 10-50 copies per cell). An EPI400/EPI300 strain is recommended. The protocol is as below:
a) Add 4 ml LB media into each test tube. Inoculate each tube with bacterial culture with antibiotic at the
proper concentration.
b) Incubate the tubes at 37°C, shaking overnight.
c) Dilute the starting culture (from step b) 1:10 into antibiotic-supplemented fresh media.
d) Supplement induction solution with a ratio of 1:1000, and grow the culture at 37°C for 4 h with vigorous
shaking (approx. 250 rpm).
e) Isolate DNA from the induced culture cells as per the protocol provided.
The pCC1BAC vector has two origins of replication – a single copy E. coli F-factor replicon (ori2 or oriS) and a high-copy origin of replication called "oriV". Initially, replication of CopyControl clones can be controlled by the F-factor replicon so the vector is present at one copy per cell. Maintaining clones at single copy ensures insert stability and allows cloning of toxic gene products. Initiation of replication from oriV requires the trfA gene product. CopyControl Vectors use a specifically engineered E. coli host strain, EPI300/EPI400, which contains a mutant trfA gene under tight control of an inducible promoter (araBAD promoter). Addition of the CopyControl Induction Solution to the growth medium induces expression of trfA and subsequent amplification of the clone to high-copy number. Induction of CopyControl BAC clones from singlecopy up to 10-20 copies per cell greatly improves the yield and purity of BAC DNA for sequencing, fingerprinting and other applications. The ParA ATPase of F plasmid, SopA, is stimulated by SopB that assembles into a partition complex on the centromere-like locus, sopC, on the plasmid cargo. Mini-F plasmid has the trans-acting genes sopA and sopB and the cis-acting site sopC which are essential for accurate partitioning of plasmid DNA molecules into both daughter cells.
- Vector Name:
- pCC1BAC
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 8139 bp
- Type:
- Cloning vector
- Replication origin:
- ori2
- Source/Author:
- EPICENTRE Biotechnologies.
- Copy Number:
- Single copy
- Growth Strain(s):
- EPI300/400
- Growth Temperature:
- 37℃
pCC1BAC vector 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
- Jang S, Liu H, Su J, Dong F, Xiong F, Liao L, Wang Y, Zhu Z. Construction and characterization of two bacterial artificial chromosome libraries of grass carp. Mar Biotechnol (NY). 2010 Jun;12(3):261-6.
pCC1BAC vector Sequence
LOCUS Exported 8139 bp DNA circular SYN 29-AUG-2024
DEFINITION Cloning vector pCC1BAC, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8139)
AUTHORS EPICENTRE Biotechnologies.
TITLE Direct Submission
JOURNAL Submitted (23-AUG-2007) 726 Post Road, Madison, WI 53713, USA
REFERENCE 2 (bases 1 to 8139)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8139)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 8139)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Submitted
(23-AUG-2007) 726 Post Road, Madison, WI 53713, USA"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8139
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 359..370
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 288..304
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 311..329
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 332..400
/label=MCS
/note="pUC18/19 multiple cloning site"
misc_feature 359..370
/label=MCS
/note="pUC18/19 multiple cloning site"
primer_bind complement(443..459)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(467..483)
/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(491..521)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(536..557)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(780..1436)
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
promoter complement(1437..1539)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
rep_origin 2397..3011
/label=oriV
/note="origin of replication for the bacterial F plasmid"
rep_origin 3087..3306
/label=ori2
/note="secondary origin of replication for the bacterial F
plasmid; also known as oriS"
CDS 3397..4149
/codon_start=1
/label=repE
/note="replication initiation protein for the bacterial F
plasmid"
/translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
misc_feature 4155..4405
/label=incC
/note="incompatibility region of the bacterial F plasmid"
CDS 4731..5903
/codon_start=1
/label=sopA
/note="partitioning protein for the bacterial F plasmid"
/translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
CDS 5906..6874
/codon_start=1
/label=sopB
/note="partitioning protein for the bacterial F plasmid"
/translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
misc_feature 6950..7423
/label=sopC
/note="centromere-like partitioning region of the bacterial
F plasmid"
misc_feature complement(7683..8081)
/label=cos
/note="lambda cos site; allows packaging into phage lambda
particles"
protein_bind complement(8099..8132)
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."