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ID   YEPLAC112  preliminary; circular DNA; SYN; 4989 BP.
XX
AC   X75458; L26353;
XX
DT   03-FEB-1994 (Rel. 8, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
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DE   Saccharomyces/E.coli plasmid vector YEplac112 - complete.
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KW   cloning vector.
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OS   Cloning vector
OC   Artificial sequences; Cloning vehicles.
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RN   [1]
RP   1-4989
RC   YEplac112
RA   Gietz R.D.;
RT   ;
RL   Submitted (13-MAY-1993) by:
RL   Gietz R.D., Health Science Ctr., University of Manitoba,
RL   Manitoba, CANADA.
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RN   [2]
RP   1-4989
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).
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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: 415330
CC   NM (YEplac112)
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)(YEplac181)(YEplac195)(YIplac128)
CC   BR (YIplac204)(YIplac211)
CC   OF ()
CC   OR ()
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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, yeast 2 micron 704..704
FT                   Klenow
FT                   -> plasmid 5937bp
FT                   1. plasmid EcoRI 5937bp
FT                   blunt end:blunt end
FT                   2. YRp7 BglII-EcoRI 852bp 604..1456, TRP1
FT                   blunt end:blunt end
FT                   -> YEplac112 4989bp"
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 TRP1 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 4989 BP; 1327 A; 1149 C; 1086 G; 1427 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 tgatctttta tgcttgcttt tcaaaaggct tgcaggcaag
     tgcacaaaca atacttaaat aaatactact cagtaataac ctatttctta gcatttttga
     cgaaatttgc tattttgtta gagtctttta caccatttgt ctccacacct ccgcttacat
     caacaccaat aacgccattt aatctaagcg catcaccaac attttctggc gtcagtccac
     cagctaacat aaaatgtaag ctctcggggc tctcttgcct tccaacccag tcagaaatcg
     agttccaatc caaaagttca cctgtcccac ctgcttctga atcaaacaag ggaataaacg
     aatgaggttt ctgtgaagct gcactgagta gtatgttgca gtcttttgga aatacgagtc
     ttttaataac tggcaaaccg aggaactctt ggtattcttg ccacgactca tctccatgca
     gttggacgat atcaatgccg taatcattga ccagagccaa aacatcctcc ttaggttgat
     tacgaaacac gccaaccaag tatttcggag tgcctgaact atttttatat gcttttacaa
     gacttgaaat tttccttgca ataaccgggt caattgttct ctttctattg ggcacacata
     taatacccag caagtcagca tcggaatcta gtgcacattc tgcggcctct gtgctctgca
     agccgcaaac tttcaccaat ggaccagaac tacctgtgaa attaataaca gacatactcc
     aagctgcctt tgtgtgctta atcacgtata ctcacgtgct caatagtcac caatgccctc
     cctcttggcc ctctcctttt cttttttcga ccgaattaat tcttgaagac gaaagggcct
     cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     agcggaaga
//