pHiL-D2
Search name
pHiL-D2,Plasmid pHiL-D2,pHiL-D2 vector
pHiL-D2 Information
Bacterial Resistance:
Promoter: AOX1
Replicator: pUC ori, FI ori
Plasmid size: 8210bp
Prokaryotic resistance: Amp
Screening markers: HIS4
Cloned strain: DH5 alpha
Culture conditions: 37 centigrade, aerobic LB
Expression host: yeast cells
Culture conditions: 28 C, YPD
5'sequencing primers: AOX5:GACTGGTTCCAATTGACAAGC
3'sequencing primers: AOX3:GGCAAATGGCATTCTGACAT
pHiL-D2 Description
1. the size of pHIL-D2 carrier is 8210 BP, which is a fusion expression vector.
In the 2. vector construction, the single restriction endonuclease loci available for EcoR I.
The protein gene of 3. intracellular expression.
4. in the process of vector construction, we need to start the gene codon ATG.
5. Pichia pastoris was screened by HIS4.
6. in order to insert the gene into the AOX region of GS115 or KM71, the SacI restrictive endonuclease linearized plasmids (His+Mut+ genotypes in GS115 and His+ MutS genotype in KM71) were used to insert the gene into GS115 or KM71 His4 regions. To produce His+ MutS genotype) insert the gene into the AOX1 region of GS115, and use Bgl II to linearize the plasmid (producing His+MutS genotype).
pHiL-D2 Sequence
LOCUS Exported 8210 bp ds-DNA circular SYN 10-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 17
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8210)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Saturday, September 10, 2016 from SnapGene Viewer 3.1.4
FEATURES Location/Qualifiers
source 1..8210
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 14..953
/gene="Pichia pastoris AOX1"
/note="AOX1 promoter"
/note="inducible promoter, regulated by methanol"
terminator 1031..1277
/gene="Pichia pastoris AOX1"
/note="AOX1 terminator"
/note="transcription terminator for AOX1"
CDS complement(1690..4224)
/codon_start=1
/gene="Pichia pastoris HIS4"
/product="multifunctional enzyme, required for histidine
biosynthesis"
/note="PpHIS4"
/note="auxotrophic marker for Pichia pastoris"
/translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE
LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL
VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII
DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI
PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER
TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED
IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP
KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE
LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVETCP
GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE
VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA
LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED
AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV
QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK
HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"
misc_feature 4579..5335
/note="AOX1 3' fragment"
/note="region downstream of Pichia pastoris AOX1 gene"
promoter 5582..5686
/gene="bla"
/note="AmpR promoter"
CDS 5687..6547
/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"
rep_origin complement(6589..7044)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
rep_origin 7155..7743
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 7929..8069
/note="bom"
/note="basis of mobility region from pBR322"
ORIGIN
1 agatcgcggc cgcgatctaa catccaaaga cgaaaggttg aatgaaacct ttttgccatc
61 cgacatccac aggtccattc tcacacataa gtgccaaacg caacaggagg ggatacacta
121 gcagcagacc gttgcaaacg caggacctcc actcctcttc tcctcaacac ccacttttgc
181 catcgaaaaa ccagcccagt tattgggctt gattggagct cgctcattcc aattccttct
241 attaggctac taacaccatg actttattag cctgtctatc ctggcccccc tggcgaggtc
301 atgtttgttt atttccgaat gcaacaagct ccgcattaca cccgaacatc actccagatg
361 agggctttct gagtgtgggg tcaaatagtt tcatgttccc aaatggccca aaactgacag
421 tttaaacgct gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa
481 gtttggttcg ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca
541 taccgtttgt cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt
601 agcgcagtct ctctatcgct tctgaacccg gtggcacctg tgccgaaacg caaatgggga
661 aacaacccgc tttttggatg attatgcatt gtcctccaca ttgtatgctt ccaagattct
721 ggtgggaata ctgctgatag cctaacgttc atgatcaaaa tttaactgtt ctaaccccta
781 cttgacaggc aatatataaa cagaaggaag ctgccctgtc ttaaaccttt ttttttatca
841 tcattattag cttactttca taattgcgac tggttccaat tgacaagctt ttgattttaa
901 cgacttttaa cgacaacttg agaagatcaa aaaacaacta attattcgaa acgaggaatt
961 cgccttagac atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc
1021 tagattctaa tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt
1081 tgatactttt ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc
1141 tcgtacgagc ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg
1201 tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta
1261 cagaagatta agtgagaagt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt
1321 ttatcacagt taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc
1381 atcgtcatcc tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta
1441 ctgccgggcc tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg
1501 ctgctagcgc tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc
1561 gaccgctttg gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac
1621 gcgatcatgg cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc
1681 tctaaataat taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc
1741 atctagacct tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc
1801 aggagttacg tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta
1861 ttgacgggca tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca
1921 actctctgga gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata
1981 agaagaagca ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc
2041 caaagcctgc tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac
2101 aatttcaacc cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag
2161 ctccttgtct gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc
2221 ttgagacaga aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag
2281 aacttcagaa ggcccagcag gcatgtcaat actacacagg gctga <
文献和实验
技术资料
