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ID   YEPLAC195  preliminary; circular DNA; SYN; 5241 BP.
XX
AC   X75459; L26355;
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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 YEplac195 - 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   1-5241
RC   YEplac195
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   1-5241
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: 415332
CC   NM (YEplac195)
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)(YEplac181)(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                   \ yeast 2 micron
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 HindIII-SmaI 37bp 6235..6272, 7213bp
FT                   2. YEp24 HindIII-SmaI 1108bp 2273..3381, URA3 gene
FT                   -> plasmid 8321bp
FT                   1. plasmid PstI 8321bp, M13mp19 6251..6251
FT                   oligo mutagenesis
FT                   -> plasmid2 8321bp
FT                   1. pIBI30 remove SmaI-HindIII 51bp 286..337, 2875bp
FT                   2. plasmid2 HindIII-SmaI 1108bp 2273..3381, URA3 gene
FT                   -> plasmid3 4000bp
FT                   1. plasmid3 HindIII-SmaI 1108bp 2273..3381, URA3 gene
FT                   blunt end:blunt end
FT                   2. plasmid4 EcoRI 5937bp 4292..4292
FT                   blunt end:blunt end
FT                   -> YEplac195 5241bp"
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 URA3 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 5241 BP; 1401 A; 1188 C; 1127 G; 1525 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 tgggtaataa ctgatataat taaattgaag ctctaatttg
     tgagtttagt atacatgcat ttacttataa tacagttttt tagttttgct ggccgcatct
     tctcaaatat gcttcccagc ctgcttttct gtaacgttca ccctctacct tagcatccct
     tccctttgca aatagtcctc ttccaacaat aataatgtca gatcctgtag agaccacatc
     atccacggtt ctatactgtt gacccaatgc gtctcccttg tcatctaaac ccacaccggg
     tgtcataatc aaccaatcgt aaccttcatc tcttccaccc atgtctcttt gagcaataaa
     gccgataaca aaatctttgt cgctcttcgc aatgtcaaca gtacccttag tatattctcc
     agtagatagg gagcccttgc atgacaattc tgctaacatc aaaaggcctc taggttcctt
     tgttacttct tctgccgcct gcttcaaacc gctaacaata cctgggccca ccacaccgtg
     tgcattcgta atgtctgccc attctgctat tctgtataca cccgcagagt actgcaattt
     gactgtatta ccaatgtcag caaattttct gtcttcgaag agtaaaaaat tgtacttggc
     ggataatgcc tttagcggct taactgtgcc ctccatggaa aaatcagtca agatatccac
     atgtgttttt agtaaacaaa ttttgggacc taatgcttca actaactcca gtaattcctt
     ggtggtacga acatccaatg aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa
     tagcttggca gcaacaggac taggatgagt agcagcacgt tccttatatg tagctttcga
     catgatttat cttcgtttcc tgcatgtttt tgttctgtgc agttgggtta agaatactgg
     gcaatttcat gtttcttcaa cactacatat gcgtatatat accaatctaa gtctgtgctc
     cttccttcgt tcttccttct gttcggagat taccgaatca aaaaaatttc aaagaaaccg
     aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaat tgaaaagcta 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
//