The PichiaPink Secreted Vector Kit contains the pPINKα-HC vector for use with the PichiaPink Yeast Expression System. The pPINKα-HC vector is built around the ADE2 gene for complementing the complementing the ade2 deletion in the PichiaPink strains. The pPINKα-HC vector has the α-mating factor secretion signal sequence so that all proteins are secreted. You can order the PichiaPink Secreted Vector Kit to refill your PichiaPink Yeast Expression System Kit.
| 载体基本信息 | |||
| 载体名称 | pPINKα-HC | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 7898 bp | 载体类型 | Yeast Plasmids |
| 载体来源 | Invitrogen (Life Technologies) | 筛选标记 | ADE2 |
| 拷贝数 | High copy number | 启动子 | AOX1 |
| 克隆方法 | Enzyme digestion and ligation | 5引物 | 5´ AOX1:5´-GACTGGTTCCAATTGACAAGC-3´ alpha-Factor-F: 5´-TACTATTGCCAGCATTGCTGC-3´ |
| 3引物 | 3´ AOX1:5´-GCAAATGGCATTCTGACATCC-3´ | ||
pPINKα-HC 载体载体图谱
pPINKα-HC 载体载体序列
LOCUS Exported 7898 bp ds-DNA circular SYN 13-SEP-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pPink-alpha-HC
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7898)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7898
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 2..940
/gene="Pichia pastoris AOX1"
/note="AOX1 promoter"
/note="inducible promoter, regulated by methanol"
terminator 1248..1497
/gene="S. cerevisiae CYC1"
/note="CYC1 terminator"
/note="transcription terminator for CYC1"
promoter 1517..1529
/gene="Pichia pastoris ADE2"
/note="PpADE2 HC promoter"
/note="weak 13-bp promoter fragment that requires high-copy
integration for efficient expression"
CDS 1530..3221
/codon_start=1
/gene="Pichia pastoris ADE2"
/product="phosphoribosylaminoimidazole carboxylase from
Pichia pastoris"
/note="PpADE2"
/note="essential for the biosynthesis of purine
nucleotides, including adenine"
/translation="MDSQVIGILGGGQLGRMIVEAASRLNIKTVILDDGFSPAKHINAA
QDHIDGSFKDEEAIAKLAAKCDVLTVEIEHVNTDALKRVQDRTGIKIYPLPETIELIKD
KYLQKEHLIKHNISVTKSQGIESNEKALLLFGEENGFPYLLKSRTMAYDGRGNFVVESK
EDISKALEFLKDRPLYAEKFAPFVKELAVMVVRSLEGEVFSYPTVETVHKDNICHIVYA
PARVNDTIQKKAQILAENTVKTFPGAGIFGVEMFLLSDGELLVNEIAPRPHNSGHYTID
ACVTSQFEAHVRAITGLPMPLDFTKLSTSNTNAIMLNVLGAEKSHGELEFCRRALETPG
ASVYLYGKTTRLARKMGHINIIGSSMLEAEQKLEYILEESTHLPSSTVSADTKPLVGVI
MGSDSDLPVISKGCDILKQFGVPFEVTIVSAHRTPQRMTRYAFEAASRGIKAIIAGAGG
AAHLPGMVAAMTPLPVIGVPVKGSTLDGVDSLHSIVQMPRGVPVATVAINNATNAALLA
IRILGTIDHKWQKEMSKYMNAMETEVLGKASNLESEGYESYLKNRL"
promoter 3448..3597
/gene="Pichia pastoris TRP2"
/note="PpTRP2 promoter"
/note="promoter for Pichia pastoris TRP2"
CDS 3598..5214
/codon_start=1
/gene="Pichia pastoris TRP2"
/product="anthranilate synthase from Pichia pastoris"
/note="PpTRP2"
/note="catalyzes the initial step of tryptophan
biosynthesis"
/translation="MSVSDSHLATDYFGTQLTKQIHPSAESFLDIVKNDRQSHSPETVP
NLYPIYKFIQSDFLTPHLAYIKLTNSSETPSPSFLLESAKNGDTVDRYTFMGHSPRKII
KTGPLEGAEVDPLVLLEKELKGTRQAQLPGIPRLSGGAIGYISYDCIKYFEPKTERKLK
DVLQLPEAALMLFDTIVAFDNVYQRFQVIGNVSLSVDDSDEAILEKYYKTREEVEKISK
VVFDNKTVPYYEQKDIIQGQTFTSNIGQEGYENHVRKLKEHILKGDIFQAVPSQRVARP
TSLHPFNIYRHLRTVNPSPYMFYIDYLDFQVVGASPELLVKSDNNNKIITHPIAGTLPR
GKTIEEDDNYAKQLKSSLKDRAEHVMLVDLARNDINRVCEPTSTTVDRLLTVERFSHVM
HLVSEVSGTLRPNKTRFDAFRSIFPAGTVSGAPKVRAMQLIGELEGEKRGVYAGAVGHW
SYDGKSMDTCIALRTMVVKDGVAYLQAGGGIVYDSDPYDEYIETMNKMRSNNNTILEAE
KIWTDRLARDENQSESEENDQ"
primer_bind complement(5276..5292)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 5300..5316
/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(5324..5354)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 5369..5390
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(5678..6266)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6437..7297)
/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"
promoter complement(7298..7402)
/gene="bla"
/note="AmpR promoter"
primer_bind 7876..7892
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
