pYES2/CT酿酒酵母质粒

基本信息

启动子:	GAL1 promoter
复制子:	2μ ori,ori,FI ori
终止子:	CYC1
质粒大小:	5963bp
原核抗性:	Amp
筛选标记:	URA3
克隆菌株:	DH5α
培养条件:	37℃，有氧，LB
表达宿主:	酵母细胞
5'测序引物:	GAL1-F: ATTTTCGGTTTGTATTACTTC
3'测序引物:	GALI-R: GTTCTTAATACTAACATAACT

质粒简介

        The pYC2/CT vector contains the CEN6/ARSH4 sequence (Sikorski and Hieter, 1989) for maintenance and replication in yeast. The CEN6/ARSH4 sequence is a 518 bp hybrid DNA fragment that contains a yeast centromere sequence (CEN) and an autonomously replicating sequence (ARS) (Sikorski and Hieter, 1989). The CEN6 sequence is derived from the CEN6 locus of yeast chromosome 6 (Panzeri and Philippsen, 1982) while the ARSH4 sequence is derived from the yeast histone H4-associated ARS (Bouton and Smith, 1986). When placed in a heterologous expression plasmid (i.e. pYC2/CT), the presence of the CEN6/ARSH4 sequence allows non-integrative centromeric maintenance and low copy number replication of the plasmid (generally 1–2 copies per cell). 

质粒图谱

质粒序列

LOCUS       Exported                5963 bp ds-DNA     circular SYN 11-SEP-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled 4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5963)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5963
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        2..443
                     /gene="S. cerevisiae GAL1"
                     /note="GAL1 promoter"
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2..119
                     /bound_moiety="Gal4"
                     /note="UAS"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     promoter        475..493
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             607..648
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /note="V5 tag"
                     /translation="GKPIPNPLLGLDST"
     CDS             658..675
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     terminator      709..956
                     /gene="S. cerevisiae CYC1"
                     /note="CYC1 terminator"
                     /note="transcription terminator for CYC1"
     rep_origin      complement(1204..1792)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1963..2823)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     CDS             complement(2919..3722)
                     /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(3723..3938)
                     /gene="S. cerevisiae URA3"
                     /note="URA3 promoter"
     rep_origin      4542..5422
                     /note="2u ori"
                     /note="yeast 2u plasmid origin of replication"
     rep_origin      complement(5491..5946)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt
       61 cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga
      121 acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac
      181 ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga
      241 ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat
      301 taacagatat ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc
      361 ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac
      421 ctctatactt taacgtcaag gagaaaaaac cccggatcgg actactagca gctgtaatac
      481 gactcactat agggaatatt aagcttggta ccgagctcgg atccactagt aacggccgcc
      541 agtgtgctgg aattctgcag atatccagca cagtggcggc cgctcgagtc tagagggccc
      601 ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgcg taccggtcat
      661 catcaccatc accattgagt ttaaacccgc tgatcctaga gggccgcatc atgtaattag
      721 ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag
      781 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
      841 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
      901 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc
      961 tgcggccctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1021 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     1081 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     1141 gaacatgtga gcaaaaggcc agcaaaagcc caggaaccgt aaaaaggccg cgttgctggc
     1201 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     1261 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     1321 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     1381 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     1441 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     1501 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     1561 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     1621 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     1681 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     1741 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     1801 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     1861 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     1921 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     1981 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     2041 cgtgtagata actacgatac gggagcgctt accatctggc cccagtgctg caatgatacc
     2101 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     2161 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     2221 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg gcattgctac
     2281 aggcatcgtg gtgtcactct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     2341 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     2401 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     2461 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     2521 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     2581 acgggataat agtgtatcac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     2641 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     2701 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     2761 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     2821 catactcttc ctttttcaat gggtaataac tgatataatt aaattgaagc tctaatttgt
     2881 gagtttagta tacatgcatt tacttataat acagtttttt agttttgctg gccgcatctt
     2941 ctcaaatatg cttcccagcc tgcttttctg taacgttcac cctctacctt agcatccctt
     3001 ccctttgcaa atagtcctct tccaacaata ataatgtcag atcctgtaga gaccacatca
     3061 tccacggttc tatactgttg acccaatgcg tctcccttgt catctaaacc cacaccgggt
     3121 gtcataatca accaatcgta accttcatct cttccaccca tgtctctttg agcaataaag
     3181 ccgataacaa aatctttgtc gctcttcgca atgtcaacag tacccttagt atattctcca
     3241 gtagataggg agcccttgca tgacaattct gctaacatca aaaggcctct aggttccttt
     3301 gttacttctt ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt
     3361 gcattcgtaa tgtctgccca ttctgctatt ctgtatacac ccgcagagta ctgcaatttg
     3421 actgtattac caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg
     3481 gataatgcct ttagcggctt aactgtgccc tccatggaaa aatcagtcaa gatatccaca
     3541 tgtgttttta gtaaacaaat tttgggacct aatgcttcaa ctaactccag taattccttg
     3601 gtggtacgaa catccaatga agcacacaag tttgtttgct tttcgtgcat gatattaaat
     3661 agcttggcag caacaggact aggatgagta gcagcacgtt ccttatatgt agctttcgac
     3721 atgatttatc ttcgtttcct gcaggttttt gttctgtgca gttgggttaa gaatactggg
     3781 caatttcatg tttcttcaac actacatatg cgtatatata ccaatctaag tctgtgctcc
     3841 ttccttcgtt cttccttctg ttcggagatt accgaatcaa aaaaatttca aagaaaccga
     3901 aatcaaaaaa aagaataaaa aaaaaatgat gaattgaatt gaaaagctag cttatcgatg
     3961 ataagctgtc aaagatgaga attaattcca cggactatag actatactag atactccgtc
     4021 tactgtacga tacacttccg ctcaggtcct tgtcctttaa cgaggcctta ccactctttt
     4081 gttactctat tgatccagct cagcaaaggc agtgtgatct aagattctat cttcgcgatg
     4141 tagtaaaact agctagaccg agaaagagac tagaaatgca aaaggcactt ctacaatggc
     4201 tgccatcatt attatccgat gtgacgctgc agcttctcaa tgatattcga atacgctttg
     4261 aggagataca gcctaatatc cgacaaactg ttttacagat ttacgatcgt acttgttacc
     4321 catcattgaa ttttgaacat ccgaacctgg gagttttccc tgaaacagat agtatatttg
     4381 aacctgtata ataatatata gtctagcgct ttacggaaga caatgtatgt atttcggttc
     4441 ctggagaaac tattgcatct attgcatagg taatcttgca cgtcgcatcc ccggttcatt
     4501 ttctgcgttt ccatcttgca cttcaatagc atatctttgt taacgaagca tctgtgcttc
     4561 attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct
     4621 gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg
     4681 cttcattttt gtaaaacaaa aatgcaacgc gacgagagcg ctaatttttc aaacaaagaa
     4741 tctgagctgc atttttacag aacagaaatg caacgcgaga gcgctatttt accaacaaag
     4801 aatctatact tcttttttgt tctacaaaaa tgcatcccga gagcgctatt tttctaacaa
     4861 agcatcttag attacttttt ttctcctttg tgcgctctat aatgcagtct cttgataact
     4921 ttttgcactg taggtccgtt aaggttagaa gaaggctact ttggtgtcta ttttctcttc
     4981 cataaaaaaa gcctgactcc acttcccgcg tttactgatt actagcgaag ctgcgggtgc
     5041 attttttcaa gataaaggca tccccgatta tattctatac cgatgtggat tgcgcatact
     5101 ttgtgaacag aaagtgatag cgttgatgat tcttcattgg tcagaaaatt atgaacggtt
     5161 tcttctattt tgtctctata tactacgtat aggaaatgtt tacattttcg tattgttttc
     5221 gattcactct atgaatagtt cttactacaa tttttttgtc taaagagtaa tactagagat
     5281 aaacataaaa aatgtagagg tcgagtttag atgcaagttc aaggagcgaa aggtggatgg
     5341 gtaggttata tagggatata gcacagagat atatagcaaa gagatacttt tgagcaatgt
     5401 ttgtggaagc ggtattcgca atgggaagct ccaccccggt tgataatcag aaaagcccca
     5461 aaaacaggaa gattgtataa gcaaatattt aaattgtaaa cgttaatatt ttgttaaaat
     5521 tcgcgttaaa tttttgttaa atcagctcat tttttaacga atagcccgaa atcggcaaaa
     5581 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttccaaca
     5641 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaagg gtctatcagg
     5701 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     5761 aagcagtaaa tcggaagggt aaacggatgc ccccatttag agcttgacgg ggaaagccgg
     5821 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggggctagg gcggtgggaa
     5881 gtgtaggggt cacgctgggc gtaaccacca cacccgccgc gcttaatggg gcgctacagg
     5941 gcgcgtgggg atgatccact agt