pPIC3.5K
Search name
pPIC3.5K,Plasmid pPIC3.5K,pPIC3.5K vector
pPIC3.5K Information
Promoter: AOX1
Replicon: pBR322 ori
Plasmid classification: yeast series, Pichia pastoris expression vector.
Plasmid size: 9004bp
Prokaryotic resistance: Amp
Screening markers: HIS4
Cloned strain: DH5 alpha
Culture conditions: 37 centigrade, aerobic LB
Expression host: yeast cells
5'sequencing primers: AOX5:GACTGGTTCCAATTGACAAGC
3'sequencing primers: AOX3:GGCAAATGGCATTCTGACAT
Use: Yeast expression
pPIC3.5K Description
PIC3.5K is a vector for identifying multiple copies of genes inserted into Pichia pastoris genome. The size of the 1. pPIC3.5K carrier is 9004 BP.
2., in the region of polyclonal sites, there are 5 single restriction sites: BamH I, Snab I, EcoR I, Avr II and Not I.
3. intracellular expression. When constructing the
4. vector, we need to include a ATG promoter and Kozak sequence. (Cavener and Stuart, 1991; Kozak, 1987; Kozak, 1990)
5. HIS4 gene screening in Pichia pastoris.
6. inserting genes into AOX1 of GS115 and KM71 requires the use of Sac I linearized plasmids (the genotype produced by GS115 is His+ Mut+, and the genotype produced by KM71 yeast is His+ MutS).
7. inserting genes into HIS4 of GS115 and KM71 requires the use of Sal I linearized plasmids (the genotype produced by GS115 is His+ Mut+, and the genotype produced by KM71 yeast is His+ MutS).
8., insert the gene into the AOX1 gene of GS115, and also use Bgl II restriction enzyme to linearize plasmid, and the yeast genotype produced is His+ MutS.
pPIC3.5K Sequence
LOCUS Exported File 9004 bp ds-DNA circular SYN 31-1-2015
DEFINITION .
ACCESSION .
VERSION .
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9004)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported 2015-1-31
FEATURES Location/Qualifiers
source 1..9004
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 1049..1295
/gene="Pichia pastoris AOX1"
/note="AOX1 terminator"
/note="transcription terminator for AOX1"
CDS complement(1708..4242)
/codon_start=1
/gene="Pichia pastoris HIS4"
/locus_tag="
"
/product="multifunctional enzyme, required for histidine
biosynthesis"
/note="PpHIS4"
/note="auxotrophic marker for Pichia pastoris"
/protein_id="
"
/translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE
LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL
VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII
DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI
PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER
TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED
IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP
KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE
LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVETCP
GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE
VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA
LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED
AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV
QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK
HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"
CDS complement(4656..5471)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
misc_feature 5850..6606
/note="AOX1 3' fragment"
/note="region downstream of Pichia pastoris AOX1 gene"
misc_feature 6749..6889
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(7075..7663)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7834..8694)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(8695..8799)
/gene="bla"
/note="AmpR promoter"
ORIGIN
1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag
61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt
121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc
181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta
241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta
301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca taccgtttgt
541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
901 caacttgaga ag
