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ID YEPLAC195 preliminary; circular DNA; SYN; 5241 BP.
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AC X75459; L26355;
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 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)"
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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
//