pABAI酵母单杂交质粒

基本信息

启动子:	URA3 promoter
复制子:	ColE1 ori
质粒分类:	酵母系列质粒；酵母杂交质粒；单杂交类质粒
质粒大小:	4894bp
原核抗性:	氨苄青霉素Amp（100μg/ml）
筛选标记:	URA3
克隆菌株:	DH5α等大肠杆菌
培养条件:	37℃，有氧，LB
表达宿主:	Y1Hgold等酵母菌
培养条件:	30℃，YPDA，有氧
5'测序引物:	pABAI-F（GTTCCTTATATGTAGCTTTCGACA）
3'测序引物:	pABAI-R（CCATCTCGAAAAAGGGTTTGCC）
备注:	低拷贝质粒

质粒简介

          pABAI质粒是一种单杂交酵母报告质粒，可以识别和分析蛋白质与DNA之间的相互作用，而不仅仅是证明蛋白之间的相互作用。这个质粒具有AbA抗性因而具有很强的筛选能力，可极大地降低背景水平。
          pABAI is a yeast reporter vector that can be used in one-hybrid assays to identify and characterize DNA-binding proteins. The vector, which was specifically designed for use with the Matchmaker Gold Yeast One-Hybrid Library Screening System, contains a multiple cloning site upstream of the yeast iso-1-cytochrome C minimal promoter(not shown) and the AUR1-C gene, an antibiotic resistance gene that confers resistance to Aureobasidin A. A cis-acting element (i.e., a target sequence) can be inserted into the MCS and used as bait to screen GAL4 AD/cDNA fusion libraries for proteins that interact with the target sequence. Positive protein-DNA (i.e.,one-hybrid) interactions drive the expression of AUR1-C. As a result, one-hybrid interactions can be detected by selecting for yeast that are resistant to AbA.
          pABAI cannot be propagated episomally in yeast; it can only be stablymaintained through integration into the host genome. Integration is accomplished via homologous recombination between the vector’s URA3 gene and the ura3-52 locus of the yeast strain provided in theMatchmaker Gold Yeast One-Hybrid System. URA3 is a nutritional marker that can also be used for selection of recombinant yeast. The vector also contains a Col E1 origin of replication and an ampicillin resistance gene (Ampr) for propagation and selection in E.coli.
          To use pAbAi in a one-hybrid assay, clone one or more copies of a cis-acting element into the MCS. To facilitate recombination of the vector with the host’s ura3-52 locus, linearize the vector with either BbsI or BstBI, then introduce the linearized vector into competent yeast cells using the protocols in the Matchmaker Gold Yeast One-Hybrid Library Screening System User Manual.
          Insertion of your target sequence may alter the basal expression of AUR1-C. Therefore, before starting a onehybrid analysis, basal levels of AUR1-C should be determined as described in the User Manual.
          •    Suitable host strains: DH5α and other general purpose strains.
          •    Selectable marker: plasmid confers resistance to ampicillin (100 µg/ml) to E. coli hosts.
          •    Copy number: low

质粒图谱

 

质粒序列

LOCUS       Exported                4894 bp ds-DNA     circular SYN 10-SEP-2016
DEFINITION  synthetic circular DNA
KEYWORDS    Untitled 21
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4894)
  TITLE     Direct Submission
  JOURNAL   Exported Saturday, September 10, 2016 from Plasmid
FEATURES             Location/Qualifiers
     source          1..4894
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             185..1390
                     /codon_start=1
                     /gene="S. cerevisiae AUR1 with a dominant F158Y mutation"
                     /product="aureobasidin A-resistant mutant of inositol 
                     phosphorylceramide synthase"
                     /note="AurR"
                     /note="confers fungal resistance to Aureobasidin A"
                     /translation="MANPFSRWFLSERPPNCHVADLETSLDPHQTLLKVQKYKPALSDW
                     VHYIFLGSIMLFVFITNPAPWIFKILFYCFLGTLFIIPATSQFFFNALPILTWVALYFT
                     SSYFPDDRRPPITVKVLPAVETILYGDNLSDILATSTNSFLDILAWLPYGLFHYGAPFV
                     VAAILFVFGPPTVLQGYAFAFGYMNLFGVIMQNVFPAAPPWYKILYGLQSANYDMHGSP
                     GGLARIDKLLGINMYTTCFSNSSVIFGAFPSLHSGCATMEALFFCYCFPKLKPLFIAYV
                     CWLWWSTMYLTHHYFVDLMAGSVLSYVIFQYTKYTHLPIVDTSLFCRWSYTSIEKYDIS
                     KSDPLAADSNDIESVPLSNLELDFDLNMTDEPSVSPSLFDGSTSVSRSSATSITSLGVK
                     RA"
     primer_bind     complement(1645..1661)
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1669..1685
                     /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(1693..1723)
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    1738..1759
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2047..2635)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2806..3666)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(3667..3771)
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             complement(3875..4678)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /note="URA3"
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4679..4894)
                     /gene="S. cerevisiae URA3"
                     /note="URA3 promoter"
ORIGIN
        1 aagcttgaat tcgagctcgg tacccgggga tctgtcgacc tcgaggcatg tgctctgtat
       61 gtatataaaa ctcttgtttt cttcttttct ctaaatattc tttccttata cattaggtcc
      121 tttgtagcat aaattactat acttctatag acacgcaaac acaaatacac acactaaatt
      181 aataatggca aacccttttt cgagatggtt tctatcagag agacctccaa actgccatgt
      241 agccgattta gaaacaagtt tagatcccca tcaaacgttg ttgaaggtgc aaaaatacaa
      301 acccgcttta agcgactggg tgcattacat cttcttggga tccatcatgc tgtttgtgtt
      361 cattactaat cccgcacctt ggatcttcaa gatccttttt tattgtttct tgggcacttt
      421 attcatcatt ccagctacgt cacagttttt cttcaatgcc ttgcccatcc taacatgggt
      481 ggcgctgtat ttcacttcat cgtactttcc agatgaccgc aggcctccta ttactgtcaa
      541 agtgttacca gcggtggaaa caattttata cggcgacaat ttaagtgata ttcttgcaac
      601 atcgacgaat tcctttttgg acattttagc atggttaccg tacggactat ttcattatgg
      661 ggccccattt gtcgttgctg ccatcttatt cgtatttggt ccaccaactg ttttgcaagg
      721 ttatgctttt gcatttggtt atatgaacct gtttggtgtt atcatgcaaa atgtctttcc
      781 agccgctccc ccatggtata aaattctcta tggattgcaa tcagccaact atgatatgca
      841 tggctcgcct ggtggattag ctagaattga taagctactc ggtattaata tgtatactac
      901 atgtttttca aattcctccg tcattttcgg tgcttttcct tcactgcatt ccgggtgtgc
      961 tactatggaa gccctgtttt tctgttattg ttttccaaaa ttgaagccct tgtttattgc
     1021 ttatgtttgc tggttatggt ggtcaactat gtatctgaca caccattatt ttgtagacct
     1081 tatggcaggt tctgtgctgt catacgttat tttccagtac acaaagtaca cacatttacc
     1141 aattgtagat acatctcttt tttgcagatg gtcatacact tcaattgaga aatacgatat
     1201 atcaaagagt gatccattgg ctgcagattc aaacgatatc gaaagtgtcc ctttgtccaa
     1261 cttggaactt gactttgatc ttaatatgac tgatgaaccc agtgtaagcc cttcgttatt
     1321 tgatggatct acttctgttt ctcgttcgtc cgccacgtct ataacgtcac taggtgtaaa
     1381 gagggcttaa ggaaatccat tatgtactat ttaaaaaaca caaacttttg gatgttcggt
     1441 ttattctttt tcttttactt ttttatcatg ggagcctact tcccgttttt cccgatttgg
     1501 ctacatgaca tcaaccatat cagcaaaagt gatacgggta ttatttttgc cgctatttct
     1561 ctgttctcgc tattattcca accgctgttt ggtctgcttt ctgacaaact cggcctcgac
     1621 tctagcgaat taattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct
     1681 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     1741 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     1801 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1861 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     1921 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     1981 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2041 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2101 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2161 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2221 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2281 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2341 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2401 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2461 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2521 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2581 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2641 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2701 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     2761 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     2821 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     2881 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     2941 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3001 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3061 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3121 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3181 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3241 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3301 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3361 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3421 aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     3481 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     3541 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     3601 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     3661 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     3721 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     3781 ccgaaaagtg ccacctgggt aataactgat ataattaaat tgaagctcta atttgtgagt
     3841 ttagtataca tgcatttact tataatacag ttttttagtt ttgctggccg catcttctca
     3901 aatatgcttc ccagcctgct tttctgtaac gttcaccctc taccttagca tcccttccct
     3961 ttgcaaatag tcctcttcca acaataataa tgtcagatcc tgtagagacc acatcatcca
     4021 cggttctata ctgttgaccc aatgcgtctc ccttgtcatc taaacccaca ccgggtgtca
     4081 taatcaacca atcgtaacct tcatctcttc cacccatgtc tctttgagca ataaagccga
     4141 taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc cttagtatat tctccagtag
     4201 atagggagcc cttgcatgac aattctgcta acatcaaaag gcctctaggt tcctttgtta
     4261 cttcttctgc cgcctgcttc aaaccgctaa caatacctgg gcccaccaca ccgtgtgcat
     4321 tcgtaatgtc tgcccattct gctattctgt atacacccgc agagtactgc aatttgactg
     4381 tattaccaat gtcagcaaat tttctgtctt cgaagagtaa aaaattgtac ttggcggata
     4441 atgcctttag cggcttaact gtgccctcca tggaaaaatc agtcaagata tccacatgtg
     4501 tttttagtaa acaaattttg ggacctaatg cttcaactaa ctccagtaat tccttggtgg
     4561 tacgaacatc caatgaagca cacaagtttg tttgcttttc gtgcatgata ttaaatagct
     4621 tggcagcaac aggactagga tgagtagcag cacgttcctt atatgtagct ttcgacatga
     4681 tttatcttcg tttcctgcag gtttttgttc tgtgcagttg ggttaagaat actgggcaat
     4741 ttcatgtttc ttcaacacta catatgcgta tatataccaa tctaagtctg tgctccttcc
     4801 ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa atttcaagga aaccgaaatc
     4861 aaaaaaaaga ataaaaaaaa aatgatgaat tgaa