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ID   YCPLAC111  preliminary; circular DNA; SYN; 6112 BP.
XX
AC   X75457; L26350;
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 YCplac111 - 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   1-6112
RC   YCplac111
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   1-6112
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
RC   [pTC1 from pT322 & CEN4]
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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RN   [3]
RC   YRpmini7 from YRp17
RC   from pNN279 & YRpmini7
RC   from pNN280 series & YRpmini7
RA   Mann C., Davis R.M.;
RT   "Structure and sequence of the centromeric DNA of chromosome 4 in
RT   Saccharomyces cerevisiae";
RL   Mol. Cell. Biol. 6:241-245(1986).
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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: 415325
CC   NM (YCplac111)
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)(YEplac112)(YEplac181)(YEplac195)(YIplac128)
CC   BR (YIplac204)(YIplac211)
CC   OF ()
CC   OR ()
XX
FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. pBR322 EcoRI 4361bp 4360..4360
FT                   2. YRp7 EcoRI-EcoRI 1454bp 2..1456, TRP1/ARS1
FT                   -> pT322 5806bp
FT                   1. pT322 ClaI 5806bp, pBR322 25..25
FT                   blunt end:blunt end
FT                   2. CEN4 PvuII-HpaI 850bp
FT                   blunt end:blunt end
FT                   -> pTC1 6650bp
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
FT                   2. plasmid2 HpaI-AccI 1600bp, LEU2 gene
FT                   -> plasmid3 4500bp
FT                   1. pBR322 ClaI 4361bp 25..25
FT                   blunt end:blunt end
FT                   2. pTC1 EcoRV-EcoRV 2300bp, ARS1/CEN4
FT                   blunt end:blunt end
FT                   -> pAC5 6700bp
FT                   1. plasmid3 HpaI-AccI 1600bp, LEU2 gene
FT                   2. pAC5 EcoRI 6700bp
FT                   -> plasmid4 8300bp
FT                   1. plasmid4 HindIII-HindIII 3700bp
FT                   mung bean exonuclease
FT                   AatII-AatII 3680bp
FT                   2. pUC19 AatII-NdeI 2437bp 185..2622
FT                   :Klenow
FT                   -> YCplac111 6112bp"
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 6112 BP; 1756 A; 1314 C; 1291 G; 1751 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 tatcgctggg ccattctcat gaagaatatc ttgaatttat
     tgtcatatta ctagttggtg tggaagtcca tatatcggtg atcaatatag tggttgacat
     gctggctagt caacattgag ccttttgatc atgcaaatat attacggtat tttacaatca
     aatatcaaac ttaactattg actttataac ttatttaggt ggtaacattc ttataaaaaa
     gaaaaaaatt actgcaaaac agtactagct tttaacttgt atcctaggtt atctatgctg
     tctcaccata gagaatatta cctatttcag aatgtatgtc catgattcgc cgggtaaata
     catataatac acaaatctgg cttaataaag tctataatat atctcataaa gaagtgctaa
     attggctagt gctatatatt tttaagaaaa tttcttttga ctaagtccat atcgactttg
     taaaagttca ctttagcata catatattac acgagccaga aattgtaact tttgcctaaa
     atcacaaatt gcaaaattta attgcttgca aaaggtcaca tgcttataat caactttttt
     aaaaatttaa aatacttttt tattttttat ttttaaacat aaatgaaata atttatttat
     tgtttatgat taccgaaaca taaaacctgc tcaagaaaaa gaaactgttt tgtccttgga
     aaaaaagcac tacctaggag cggccaaaat gccgaggctt tcatagctta aactctttac
     agaaaatagg cattatagat cagttcgagt tttcttattc ttccttccgg ttttatcgtc
     acagttttac agtaaataag tatcacctct tagagttcga tgataagctg tcaaacatga
     gaattaattc cacatgttaa aatagtgaag gagcatgttc ggcacacagt ggaccgaacg
     tggggtaagt gcactagggt ccggttaaac ggatctcgca ttgatgaggc aacgctaatt
     atcaacatat agattgttat ctatctgcat gaacacgaaa tctttacttg acgacttgag
     gctgatggtg tttatgcaaa gaaaccactg tgtttaatat gtgtcactgt ttgatattac
     tgtcagcgta gaagataata gtaaaagcgg ttaataagtg tatttgagat aagtgtgata
     aagtttttac agcgaaaaga cgataaatac aagaaaatga ttacgaggat acggagagag
     gtatgtacat gtgtatttat atactaagct gccggcggtt gtttgcaaga ccgagaaaag
     gctagcaaga atcgggtcat tgtagcgtat gcgcctgtga acattctctt caacaagttt
     gattccattg cggtgaaatg gtaaaagtca accccctgcg atgtatattt tcctgtacaa
     tcaatcaaaa agccaaatga tttagcatta tctttacatc ttgttatttt acagatttta
     tgtttagatc ttttatgctt gcttttcaaa aggcttgcag gcaagtgcac aaacaatact
     taaataaata ctactcagta ataacctatt tcttagcatt tttgacgaaa tttgctattt
     tgttagagtc ttttacacca tttgtctcca cacctccgct tacatcaaca ccaataacgc
     catttaatct aagcgcatca ccaacatttt ctggcgtcag tccaccagct aacataaaat
     gtaagctctc ggggctctct tgccttccaa cccagtcaga aatcgagttc caatccaaaa
     gttcacctgt cccacctgct tctgaatcaa acaagggaat aaacgaatga ggtttctgtg
     aagctgcact gagtagtatg ttgcagtctt ttggaaatac gagtctttta ataactggca
     aaccgaggaa ctcttggtat tcttgccacg actcatctcc atgcagttgg acgatcgatg
     ataagctgtc aaacatgaga attaattcta ccctatgaac atattccatt ttgtaatttc
     gtgtcgtttc tattatgaat ttcatttata aagtttatgt acaaatatca taaaaaaaga
     gaatcttttt aagcaaggat tttcttaact tcttcggcga cagcatcacc gacttcggtg
     gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac ctttttaact
     gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa catcattgca
     gcagacaaga tagtggcgat agggtcaacc ttattctttg gcaaatctgg agcagaaccg
     tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc caaggacgca
     gatggcaaca aacccaagga acctgggata acggaggctt catcggagat gatatcacca
     aacatgttgc tggtgattat aataccattt aggtgggttg ggttcttaac taggatcatg
     gcggcagaat caatcaattg atgttgaacc ttcaatgtag gaaattcgtt cttgatggtt
     tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt atccaaggac
     caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct tgtgattctt
     tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc atcgtcttcc
     tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa gtcagtacct
     ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc aaagttacat
     ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc ttgttcaggt
     ctaacactac ctgtacccca tttaggacca cccacagcac ctaacaaaac ggcatcaacc
     ttcttggagg cttccagcgc ctcatctgga agtgggacac ctgtagcatc gatagcagca
     ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc agaaatagct
     ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc tggcaaaacg
     acgatcttct taggggcaga cataggggca gacattagaa tggtatatcc ttgaaatata
     tatatatatt gctgaaatgt aaaaggtaag aaaagttaga aagtaagacg attgctaacc
     acctattgga aaaaacaata ggtccttaaa taatattgtc aacttcaagt attgtgatgc
     aagcatttag tcatgaacgc ttctctattc tatatgaaaa gccggttccg gcctctcacc
     tttccttttt ctcccaattt ttcagttgaa aaaggtatat gcgtcaggcg acctctgaaa
     ttaacaaaaa atttccagtc atcgaatttg attctgtgcg atagcgcccc tgtgtgttct
     cgttatgttg aggaaaaaaa taatggttgc taagagattc gaactcttgc atcttacgat
     acctgagtat tcccacagtt aattcttgaa gacgaaaggg cctcgtgata cgcctatttt
     tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa ga
//