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ID YIPLAC128 preliminary; circular DNA; SYN; 4302 BP.
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AC X75463; L26356;
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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 YIplac128 - 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 1-4302
RC YIplac128
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 1-4302
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: 415333
CC NM (YIplac128)
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)(YEplac195)
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. M13mp19 remove BglI-BglII 498bp 6438..6936,
FT \ 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. pUC19 EcoO109 2686bp 2676..2676
FT blunt end:blunt end
FT -> YIplac128 4302bp"
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 4302 BP; 1141 A; 1027 C; 993 G; 1141 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 tggtgcactc tcagtacaat ctgctctgat gccgcatagt
taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
caccgtcatc accgaaacgc gcgagacgaa agggcctacc 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 ctgtgatttc ttgaccaacg tggtcacctg
gcaaaacgac gatcttctta ggggcagaca taggggcaga cattagaatg gtatatcctt
gaaatatata tatatattgc tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat
tgctaaccac ctattggaaa aaacaatagg tccttaaata atattgtcaa cttcaagtat
tgtgatgcaa gcatttagtc atgaacgctt ctctattcta tatgaaaagc cggttccggc
ctctcacctt tcctttttct cccaattttt cagttgaaaa aggtatatgc gtcaggcgac
ctctgaaatt aacaaaaaat ttccagtcat cgaatttgat tctgtgcgat agcgcccctg
tgtgttctcg ttatgttgag gaaaaaaata atggttgcta agagattcga actcttgcat
cttacgatac ctgagtattc ccacagttgg 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
//