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ID   YCPLAC33   preliminary; circular DNA; SYN; 5603 BP.
XX
AC   X75456; L26352;
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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 YCplac33 - 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   1-5603
RC   YCplac33
RA   Gietz R.D.;
RT   ;
RL   Submitted (13-MAY-1993) by:
RL   Gietz R.D., Health Sciuence Ctr., University of Manitoba,
RL   Manitoba, CANADA.
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RN   [2]
RP   1-5603
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: 415329
CC   NM (YCplac33)
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)(YCplac111)(YEplac112)(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. 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 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. 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 HindIII-SmaI 1108bp 2273..3381, URA3 gene
FT                   2. pAC5 EcoRI 6700bp
FT                   -> plasmid4 7800bp
FT                   1. plasmid4 HindIII-HindIII 3200bp
FT                   mung bean exonuclease
FT                   AatII-AatII 3200bp
FT                   2. pUC19 AatII-NdeI 2437bp 185..2622
FT                   :Klenow
FT                   -> YCplac111 5603bp"
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 5603 BP; 1586 A; 1232 C; 1156 G; 1629 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 attgggtaat aactgatata attaaattga agctctaatt
     tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat
     cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc
     cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca
     tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg
     ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata
     aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct
     ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc
     tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg
     tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat
     ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg
     gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc
     acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc
     ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta
     aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc
     gacatgattt atcttcgttt cctgcatgtt tttgttctgt gcagttgggt taagaatact
     gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc
     tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaagaaac
     cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga attgaaaagc taattcttga
     agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     tcagtgagcg aggaagcgga aga
//