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ID YEPLAC181 preliminary; circular DNA; SYN; 5741 BP.
XX
AC X75460; L26354;
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 YEplac181 - complete.
XX
KW cloning vector.
XX
OS Cloning vector
OC Artificial sequences; Cloning vehicles.
XX
RN [1]
RP 1-5741
RC YEplac181
RA Gietz R.D.;
RT ;
RL Submitted (13-MAY-1993) by:
RL Gietz R.D., Health Science Ctr., University of Manitoba,
RL Manitoba, CANADA.
XX
RN [2]
RP 1-5741
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: 415331
CC NM (YEplac181)
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)(YEplac195)(YIplac128)
CC BR (YIplac204)(YIplac211)
CC OF ()
CC OR ()
XX
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, YEp24 704..704
FT Klenow
FT -> plasmid4 5937bp
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 302..302
FT 2. plasmid2 HpaI-AccI 1600bp, LEU2 gene
FT -> plasmid3 4500bp
FT 1. plasmid3 HpaI-AccI 1600bp, LEU2 gene
FT blunt end:blunt end
FT 2. plasmid4 EcoRI 5937bp 4292..4292
FT blunt end:blunt end
FT -> YEplac181 5741bp"
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 5741 BP; 1568 A; 1270 C; 1260 G; 1643 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 taattctacc ctatgaacat attccatttt gtaatttcgt
gtcgtttcta ttatgaattt catttataaa gtttatgtac aaatatcata aaaaaagaga
atctttttaa gcaaggattt tcttaacttc ttcggcgaca gcatcaccga cttcggtggt
actgttggaa ccacctaaat caccagttct gatacctgca tccaaaacct ttttaactgc
atcttcaatg gccttacctt cttcaggcaa gttcaatgac aatttcaaca tcattgcagc
agacaagata gtggcgatag ggtcaacctt attctttggc aaatctggag cagaaccgtg
gcatggttcg tacaaaccaa atgcggtgtt cttgtctggc aaagaggcca aggacgcaga
tggcaacaaa cccaaggaac ctgggataac ggaggcttca tcggagatga tatcaccaaa
catgttgctg gtgattataa taccatttag gtgggttggg ttcttaacta ggatcatggc
ggcagaatca atcaattgat gttgaacctt caatgtagga aattcgttct tgatggtttc
ctccacagtt tttctccata atcttgaaga ggccaaaaca ttagctttat ccaaggacca
aataggcaat ggtggctcat gttgtagggc catgaaagcg gccattcttg tgattctttg
cacttctgga acggtgtatt gttcactatc ccaagcgaca ccatcaccat cgtcttcctt
tctcttacca aagtaaatac ctcccactaa ttctctgaca acaacgaagt cagtaccttt
agcaaattgt ggcttgattg gagataagtc taaaagagag tcggatgcaa agttacatgg
tcttaagttg gcgtacaatt gaagttcttt acggattttt agtaaacctt gttcaggtct
aacactacct gtaccccatt taggaccacc cacagcacct aacaaaacgg catcaacctt
cttggaggct tccagcgcct catctggaag tgggacacct gtagcatcga tagcagcacc
accaattaaa tgattttcga aatcgaactt gacattggaa cgaacatcag aaatagcttt
aagaacctta atggcttcgg ctgtgatctt gaccaacgtg gtcacctggc aaaacgacga
tcttcttagg ggcagacata ggggcagaca ttagaatgta tatccttgaa atatatatat
atattgctga aatgtaaaag gtaagaaaag ttagaataag acgattgcta accacctatt
ggaaaaaaca ataggtcctt aaataatatt gtcaacttca agtattgtga tgcaagcatt
tagtcatgaa cgcttctcta ttctatatga aaagccggtt ccggcctctc acctttcctt
tttctcccaa tttttcagtt gaaaaaggta tatgcgcggc gacctctgaa attaacaaaa
aatttccagt catcgaattt gtctgtgcga tagcgcccct gtgtgttctc gttatgttga
ggaaaaaaat aatggttgct aagagattcg aactcttgca tcttacgata cctgagtatt
cccacagtta 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
//