back Return to this vector's summary.
ID   YEPLAC181  preliminary; circular DNA; SYN; 5741 BP.
XX
AC   X75460; L26354;
XX
DT   03-FEB-1994 (Rel. 8, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
XX
DE   Saccharomyces/E.coli plasmid vector YEplac181 - complete.
XX
KW   cloning vector.
XX
OS   Cloning vector
OC   Artificial sequences; Cloning vehicles.
XX
RN   [1]
RP   1-5741
RC   YEplac181
RA   Gietz R.D.;
RT   ;
RL   Submitted (13-MAY-1993) by:
RL   Gietz R.D., Health Science Ctr., University of Manitoba,
RL   Manitoba, CANADA.
XX
RN   [2]
RP   1-5741
RC   plasmid1 from pIBI30 & M13mp19 & YEp24, URA3 gene
RC   plasmid2 from pIBI30 & M13mp19 & YRp7, TRP1/ARS1 gene
RC   plasmid3 from pIBI30 & M13mp19 & YEp213, LEU2 gene
RC   YCplac22 from pUC19 & pTC1
RC   pAC5 from pBR322 & pTC1
RC   YCplac33 from plasmid1 & pAC5
RC   YCplac111 from plasmid3 & pAC5
RC   p2micron19 from pBR322 & YEp24
RC   YEplac112 from p2micron19 & plasmid1
RC   YEplac181 from p2micron19 & plasmid2
RC   YEplac195 from p2micron19 & plasmid3
RC   YIplac128 from pUC19 & plasmid1
RC   YIplac204 from pUC19 & plasmid2
RC   YIplac211 from pUC19 & plasmid3
RA   Gietz R.D., Sugino A.;
RT   "New yeast-Escherichia coli shuttle vectors constructed with in
RT   vitro mutagenized yeast genes lacking six-base pair restriction
RT   sites";
RL   Gene 74:527-534(1988).
XX
CC   NOTE: Information taken from figure 2, citation [2].
CC   In E. coli plasmid pBR322, insert yeast genes into either EcoRI or
CC   Cla I using PolIK and dNTPs to fill in overhangs and T4 DNA ligase
CC   for blunt-end ligation.
CC   Any restriction site regenerated by this process was destroyed
CC   following digestion with the appropriate restriction enzyme,
CC   using PolIK and dNTPs to fill in overhangs and T4 DNA ligase to
CC   ligate blunt ends.
CC   Once the constructions were verified, the yeast genes were moved into
CC   the plasmid vector pUC19.
CC   Excise the gene from pBR322 by HindIII digestion, followed by
CC   mung-bean exonuclease treatment to produce blunt ends.
CC   Construct YCplac vectors (ARS1-CEN4) by blunt-end ligation of the
CC   1.4-kb EcoRI fragment (TRP1 & ARS1 genes) [3] into pBR322 EcoRI
CC   site (to make pT322) and subsequent blunt-end ligation of the
CC   functional 850-bp PvuII-HpaI CEN4 fragment [4] into pT322 ClaI
CC   (to make pTC1).
CC   Digest with AatII, and isolate the fragment containing yeast DNA
CC   from agarose gels and ligate to the large AatII-Nde I fragment of
CC   pUC19, which had the NdeI end filled in with PolIK (to make YCplac22).
CC   Make the other YCplac vectors by blunt-end ligation of the EcoRV
CC   fragment from pTC1, containing the ARS1 and CEN4 sequences, into ClaI
CC   pBR322 ClaI (to make pAC5).
CC   Insert the 1.1-kb HindIII-SmaI fragment (containing URA3)
CC   or the 1.6-kb HpaI-AccI fragment (containing LEU2) at pAC5 EcoRI
CC   as described above.
CC   Move these constructs into pUC19 as described above, to make
CC   YCplac33 and YCplac111, respectively.
CC   Construct the YEplac vector series (with a yeast 2 mu origin)
CC   by blunt-end ligation of the 1.5-kb SauIII fragment
CC   from YEp24, which contains the yeast 2 mu origin [5], into pBR322
CC   ClaI (to make p2mu19).
CC   Remove p2mu19 XbaI site by filling in with PolIK after digestion and
CC   blunt-end religation of this site.
CC   Ligate the three yeast genes into p2mu19 EcoRI. The fragments used
CC   for the LEU2 and the URA3 constructs were the same as those used in
CC   the YCplac constructions. However, the YEplac112 vector (TRP1)
CC   contains the 850-bp EcoRI-BglII fragment of the TRP1 gene which does
CC   not contain ARS1 function.
CC   Construct the YIplac vectors by ligating each yeast gene fragment
CC   used in YEplac vector constructions into the pUC19 EcoO109 site
CC   using PolIK to fill the sticky ends.
CC   NCBI gi: 415331
CC   NM (YEplac181)
CC   CM (yes)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (plasmid)
CC   SP ()
CC   HO (E.coli)(Saccharomyces cerevisiae)
CC   CP ()
CC   FN (cloning)(shuttle)
CC   SE ()
CC   PA (pUC19)(pBR322)(yeast 2-micron plasmid)
CC   BR (YCplac22)(YCplac33)(YCplac111)(YEplac112)(YEplac195)(YIplac128)
CC   BR (YIplac204)(YIplac211)
CC   OF ()
CC   OR ()
XX
FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. YEp24 Sau3A/MboI-Sau3A/MboI 1576bp
FT                   \ 344..1920, yeast 2 micron ori
FT                   blunt end:blunt end
FT                   2. pBR322 ClaI 4361bp 25..25
FT                   blunt end:blunt end
FT                   -> p2micron19 5937bp
FT                   1. p2micron19 XbaI 5937bp, YEp24 704..704
FT                   Klenow
FT                   -> plasmid4 5937bp
FT                   1. M13mp19 remove BglI-BglII 498bp 6438..6936, 6752bp
FT                   2. YEp213 BglI-BglII 1442bp 3492..4934, LEU2 gene
FT                   -> plasmid 8194bp
FT                   1. plasmid KpnI 8194bp, M13mp19 6278..6278
FT                   oligo mutagenesis
FT                   EcoRI 8194bp, M13mp19 6286..6286
FT                   oligo mutagenesis
FT                   -> plasmid2 8194bp
FT                   1. pIBI30 AccI 2926bp 302..302
FT                   2. plasmid2 HpaI-AccI 1600bp, LEU2 gene
FT                   -> plasmid3 4500bp
FT                   1. plasmid3 HpaI-AccI 1600bp, LEU2 gene
FT                   blunt end:blunt end
FT                   2. plasmid4 EcoRI 5937bp 4292..4292
FT                   blunt end:blunt end
FT                   -> YEplac181 5741bp"
FT   misc_feature    0..0
FT                   /note="from pBR322"
FT   misc_feature    0..0
FT                   /note="from pUC19"
FT   misc_feature    0..0
FT                   /note="poly dG-dC tail"
FT   CDS             0..0
FT                   /note="ANT yeast LEU2 gene"
FT   misc_feature    0..0
FT                   /note="poly dG-dC tail"
FT   misc_feature    0..0
FT                   /note="yeast centromere CEN4"
FT   misc_binding    0..0
FT                   /note="MCS from pUC19"
FT   rep_origin      0..0
FT                   /note="ORI E. coli pMB1 (ColE1 and pBR322)"
FT   rep_origin      0..0
FT                   /note="ORI yeast ARS1"
FT   CDS             0..0
FT                   /note="ANT E. coli beta-lactamase gene (bla)
FT                   ampicillin resistance gene (apr/amp)"
FT   CDS             0..0
FT                   /note="ANT E. coli tetracycline resistance gene (tet)"
XX
SQ   Sequence 5741 BP; 1568 A; 1270 C; 1260 G; 1643 T; 0 other;
     gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
     atgcctgcag gtcgactcta gaggatcccc gggtaccgag ctcgaattca ctggccgtcg
     ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     gcggtatttc acaccgcata tatcggatcg tacttgttac ccatcattga attttgaaca
     tccgaacctg ggagttttcc ctgaaacaga tagtatattt gaacctgtat aataatatat
     agtctagcgc tttacggaag acaatgtatg tatttcggtt cctggagaaa ctattgcatc
     tattgcatag gtaatcttgc acgtcgcatc cccggttcat tttctgcgtt tccatcttgc
     acttcaatag catatctttg ttaacgaagc atctgtgctt cattttgtag aacaaaaatg
     caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa
     atgcaacgcg aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa
     aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa
     cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc
     tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt
     ctcctttgtg cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa
     ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac
     ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc
     cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg
     ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata
     ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct
     tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc
     gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc
     acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat
     attttagtag ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag
     cgcttttggt tttcaaaagc gctctgaagt tcctatactt tctagctaga gaataggaac
     ttcggaatag gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag
     ctgcgcacat acagctcact gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat
     atatacatga gaagaacggc atagtgcgtg tttatgctta aatgcgtact tatatgcgtc
     tatttatgta ggatgaaagg tagtctagta cctcctgtga tattatccca ttccatgcgg
     ggtatcgtat gcttccttca gcactaccct ttagctgttc tatatgctgc cactcctcaa
     ttggattagt ctcatccttc aatgctatca tttcctttga tattggatcg atccgatgat
     aagctgtcaa acatgagaat taattctacc ctatgaacat attccatttt gtaatttcgt
     gtcgtttcta ttatgaattt catttataaa gtttatgtac aaatatcata aaaaaagaga
     atctttttaa gcaaggattt tcttaacttc ttcggcgaca gcatcaccga cttcggtggt
     actgttggaa ccacctaaat caccagttct gatacctgca tccaaaacct ttttaactgc
     atcttcaatg gccttacctt cttcaggcaa gttcaatgac aatttcaaca tcattgcagc
     agacaagata gtggcgatag ggtcaacctt attctttggc aaatctggag cagaaccgtg
     gcatggttcg tacaaaccaa atgcggtgtt cttgtctggc aaagaggcca aggacgcaga
     tggcaacaaa cccaaggaac ctgggataac ggaggcttca tcggagatga tatcaccaaa
     catgttgctg gtgattataa taccatttag gtgggttggg ttcttaacta ggatcatggc
     ggcagaatca atcaattgat gttgaacctt caatgtagga aattcgttct tgatggtttc
     ctccacagtt tttctccata atcttgaaga ggccaaaaca ttagctttat ccaaggacca
     aataggcaat ggtggctcat gttgtagggc catgaaagcg gccattcttg tgattctttg
     cacttctgga acggtgtatt gttcactatc ccaagcgaca ccatcaccat cgtcttcctt
     tctcttacca aagtaaatac ctcccactaa ttctctgaca acaacgaagt cagtaccttt
     agcaaattgt ggcttgattg gagataagtc taaaagagag tcggatgcaa agttacatgg
     tcttaagttg gcgtacaatt gaagttcttt acggattttt agtaaacctt gttcaggtct
     aacactacct gtaccccatt taggaccacc cacagcacct aacaaaacgg catcaacctt
     cttggaggct tccagcgcct catctggaag tgggacacct gtagcatcga tagcagcacc
     accaattaaa tgattttcga aatcgaactt gacattggaa cgaacatcag aaatagcttt
     aagaacctta atggcttcgg ctgtgatctt gaccaacgtg gtcacctggc aaaacgacga
     tcttcttagg ggcagacata ggggcagaca ttagaatgta tatccttgaa atatatatat
     atattgctga aatgtaaaag gtaagaaaag ttagaataag acgattgcta accacctatt
     ggaaaaaaca ataggtcctt aaataatatt gtcaacttca agtattgtga tgcaagcatt
     tagtcatgaa cgcttctcta ttctatatga aaagccggtt ccggcctctc acctttcctt
     tttctcccaa tttttcagtt gaaaaaggta tatgcgcggc gacctctgaa attaacaaaa
     aatttccagt catcgaattt gtctgtgcga tagcgcccct gtgtgttctc gttatgttga
     ggaaaaaaat aatggttgct aagagattcg aactcttgca tcttacgata cctgagtatt
     cccacagtta attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     acgaccgagc gcagcgagtc agtgagcgag gaagcggaag a
//