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ID YEPLAC112 preliminary; circular DNA; SYN; 4989 BP.
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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)"
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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
//