| 质粒名称: | pKSCTMF |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 85073 |
| 存储实验室: | Vladislav Verkhusha |
|
载体骨架基本信息 |
|
| 载体名称: | pBluescript II KS(-) |
| 载体出品公司: | Stratagene |
| 空载体大小: | 2900bp |
| 完整质粒大小: | 6108 bp |
| 载体修饰: | Gene insertion/deletion in Pichia pastoris |
| 载体类型: | 细菌细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | AOX1promoter - mazF - AOX1 tt- Zeocin R gene |
| 物种来源: | E. coli |
| 插入大小: | 3000 bp |
| GenBank编号 | mazF NP_417262.1 |
| 启动子: | CMV |
| 标签/融合蛋白: | Restriction Enzyme |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | EcoRV (unknown if destroyed) |
| 3’克隆位点: | SmaI (unknown if destroyed) |
| 5’测序引物 | T7 and 5 AOX1 |
| 3’测序引物 | T3 and 3AOX1 |
|
文献引用方法 |
|
| 材料和方法部分: | pKSCTMF was a gift from Sheng Yang (Addgene plasmid # 85073 ; http://n2t.net/addgene:85073 ; RRID:Addgene_85073) |
| 参考文献部分: | mazF as a counter-selectable marker for unmarked genetic modification of Pichia pastoris. Yang J, Jiang W, Yang S. FEMS Yeast Res. 2009 Jun;9(4):600-9. doi: 10.1111/j.1567-1364.2009.00503.x. Epub 2009 Mar 25. 10.1111/j.1567-1364.2009.00503.x PubMed 19416369 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pKSCTMF 6180 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Gene insertion/deletion in Pichia pastoris.
ACCESSION .
VERSION .
KEYWORDS pKSCTMF
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6180)
AUTHORS Yang J, Jiang W, Yang S
TITLE mazF as a counter-selectable marker for unmarked genetic
modification of Pichia pastoris.
JOURNAL FEMS Yeast Res. 2009 Jun;9(4):600-9. doi:
10.1111/j.1567-1364.2009.00503.x. Epub 2009 Mar 25.
PUBMED 19416369
REFERENCE 2 (bases 1 to 6180)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6180
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 37..56
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 290..307
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 424..445
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 460..490
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 498..514
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
primer_bind 503..525
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 522..538
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 522..538
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 557..577
/label=T3
/note="T3 promoter, forward primer"
promoter 559..577
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind 606..622
/label=pBluescriptKS
/note="For pBluescript vector"
primer_bind 607..623
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
terminator 704..951
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
promoter 989..1927
/gene="Pichia pastoris AOX1"
/label=AOX1 promoter
/note="inducible promoter, regulated by methanol"
CDS 2293..2322
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 2338..2355
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
terminator 2435..2681
/gene="Pichia pastoris AOX1"
/label=AOX1 terminator
/note="transcription terminator for AOX1"
promoter 2688..3095
/gene="S. cerevisiae TEF1"
/label=TEF1 promoter
/note="promoter for EF-1-alpha"
promoter 3102..3149
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 3168..3542
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
terminator 3608..3855
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
primer_bind complement(3876..3892)
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3876..3892)
/label=pBluescriptSK
/note="For pBluescript vector"
primer_bind complement(3924..3943)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(3925..3943)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3953..3970)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(3953..3969)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3962..3984)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 4111..4566
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4198..4217)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4408..4429
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 4592..4696
/gene="bla"
/label=AmpR promoter
CDS 4697..5557
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(4915..4934)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 5728..136
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
61 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
121 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
181 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
241 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
301 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
361 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
421 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgctcccggc
481 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
541 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg gtaccgggcc
601 ccccctcgag gtcgacggta tcgataagct tgattgacac gtccgacggc ggcccgacgg
661 gtccgaggcc tcggagatcc gtcccccttt tcctttgtcg atatcatgta attagttatg
721 tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga
781 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta
841 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata
901 ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg caagctggag
961 accaacaatc gaattcctgc agccctcaga tctaacatcc aaagacgaaa ggttgaatga
1021 aacctttttg ccatccgaca tccacaggtc cattctcaca cataagtgcc aaacgcaaca
1081 ggaggggata cactagcagc agaccgttgc aaacgcagga cctccactcc tcttctcctc
1141 aacacccact tttgccatcg aaaaaccagc ccagttattg ggcttgattg gagctcgctc
1201 attccaattc cttctattag gctactaaca ccatgacttt attagcctgt ctatcctggc
1261 ccccctggcg aggttcatgt ttgtttattt ccgaatgcaa caagctccgc attacacccg
1321 aacatcactc cagatgaggg ctttctgagt gtggggtcaa atagtttcat gttccccaaa
1381 tggcccaaaa ctgacagttt aaacgctgtc ttggaaccta atatgacaaa agcgtgatct
1441 catccaagat gaactaagtt tggttcgttg aaatgctaac ggccagttgg tcaaaaagaa
1501 acttccaaaa gtcggcatac cgtttgtctt gtttggtatt gattgacgaa tgctcaaaaa
1561 taatctcatt aatgcttagc gcagtctctc tatcgcttct gaaccccggt gcacctgtgc
1621 cgaaacgcaa atggggaaac acccgctttt tggatgatta tgcattgtct ccacattgta
1681 tgcttccaag attctggtgg gaatactgct gatagcctaa cgttcatgat caaaatttaa
1741 ctgttctaac ccctacttga cagcaatata taaacagaag gaagctgccc tgtcttaaac
1801 cttttttttt atcatcatta ttagcttact ttcataattg cgactggttc caattgacaa
1861 gcttttgatt ttaacgactt ttaacgacaa cttgagaaga tcaaaaaaca actaattatt
1921 cgatacgatg gtaagccgat acgtacccga tatgggcgat ctgatttggg ttgattttga
1981 cccgacaaaa ggtagcgagc aagctggaca tcgtccagct gttgtcctga gtcctttcat
2041 gtacaacaac aaaacaggta tgtgtctgtg tgttccttgt acaacgcaat caaaaggata
2101 tccgttcgaa gttgttttat ccggtcagga acgtgatggc gtagcgttag ctgatcaggt
2161 aaaaagtatc gcctggcggg caagaggagc aacgaagaaa ggaacagttg ccccagagga
2221 attacaactc attaaagcca aaattaacgt actgattggg taggtcgagc cgcggcggcc
2281 gccagctttc tagaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat
2341 catcatcatc atcattgagt ttgtagcctt agacatgact gttcctcagt tcaagttggg
2401 cacttacgag aagaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg
2461 cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga
2521 ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc tatctcgcag
2581 ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg tttttcttgg
2641 tatttcccac tcctcttcag agtacagaag attaagtgag agatccccca cacaccatag
2701 cttcaaaatg tttctactcc ttttttactc ttccagattt tctcggactc cgcgcatcgc
2761 cgtaccactt caaaacaccc aagcacagca tactaaattt cccctctttc ttcctctagg
2821 gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc ctcgtttctt
2881 tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt tctttttctt gaaaattttt
2941 ttttttgatt tttttctctt tcgatgacct cccattgata tttaagttaa taaacggtct
3001 tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg
3061 ctcattagaa agaaagcata gcaatctaat ctaagggcgg tgttgacaat taatcatcgg
3121 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga
3181 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
3241 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
3301 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
3361 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
3421 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
3481 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
3541 gacacgtccg acgcggcccg acgggtccga ggcctcggag atccgtcccc cttttccttt
3601 gtcgatatca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct
3661 ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata
3721 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga
3781 cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg
3841 aaggctttaa tttgcaagct ggagaccaac agggggatcc actagttcta gagcggccgc
3901 caccgcggtg gagctccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
3961 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
4021 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
4081 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
4141 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
4201 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
4261 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
4321 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
4381 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
4441 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
4501 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
4561 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
4621 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
4681 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
4741 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
4801 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
4861 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
4921 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
4981 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
5041 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
5101 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
5161 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
5221 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
5281 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
5341 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
5401 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
5461 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
5521 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
5581 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
5641 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
5701 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
5761 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
5821 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
5881 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
5941 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
6001 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
6061 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
6121 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
//
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