PiggyBac Dual promoter (PB513B-1)
Catalog No. PVT10876
Packing 2ug
Function Mammal Editing plasmids
PiggyBac Dual promoter (PB513B-1) Informaiton
Promoter: CMV promoter
Replicator: pUC ori, F1 ori
Terminator: SV40 poly (A) signal
Plasmid classification: lactation serial plasmids, lactation editing plasmids, suckling transposable plasmid.
Plasmid size: 7258bp
Prokaryotic resistance: ampicillin Amp (100 u g/ml)
Screening markers: green fluorescent protein CopGFP and puromycin Puro (10ug/ml).
Cloned strains of Escherichia coli, DH5 A and other Escherichia coli
Culture conditions: 37 centigrade, aerobic LB
Expression host: mammalian cells such as 293T
Culture conditions: 37 C, 5%CO2
Induction mode: no induction, instantaneous expression
5'sequencing primers: CMV-F (CGCAAATGGGCGGTAGGCGTG)
3'sequencing primers: Sv40-polyA-R (GAAATTTGTGATGCTATTGC)
PiggyBac Dual promoter (PB513B-1) Decription
PiggyBac Dual promoter (PB513B-1) is a plasmid for a mammalian cell transposing experiment, and a polyclonal site (MCS) located downstream of the CMV promoter, making the target gene or microRNA more convenient to clone. The downstream EF-1 alpha core promoter starts the expression of GFP.
The PiggyBac (PB) transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites. The powerful activity of the piggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes.
The unique features of piggyBac transposons are that there is NO Cargo Limit and it is also Reversible. Genomes containing an inserted piggyBac vector can be transiently re-transfected with the PB transposase expression vector. The PB transposase will remove the transposons from the genome, footprint-free.The Super PiggyBac transposase transient expression vector and PB513B-1 were co-transfected into HeLa cells and puromycin selection applied for 10 days (10ug/ml). Cells efficiently transposed were Puro resistant and GFP positive.
PiggyBac Dual promoter (PB513B-1) Sequence
LOCUS Exported File 7258 bp ds-DNA circular SYN 24-5-2015
KEYWORDS PiggyBac Dual promoter(PB513B-1)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7258)
TITLE Direct Submission
JOURNAL Exported 2015-5-24
FEATURES Location/Qualifiers
source 1..7258
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter complement(1..105)
/gene="bla"
/note="AmpR promoter"
rep_origin complement(131..586)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 728..744
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 1442..1645
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 3068..3121
/codon_start=1
/product="2A peptide from?Thosea asigna virus capsid
protein"
/note="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/protein_id="
"
/translation="EGRGSLLTCGDVEENPGP"
CDS 3122..3721
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
polyA_signal 3829..3950
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(5237..5253)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 5261..5277
/bound_moiety="lac repressor encoded by lacI"
/note="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(5285..5315)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 5330..5351
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(5639..6227)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6398..7258)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
ORIGIN
1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc