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ID YCPLAC111 preliminary; circular DNA; SYN; 6112 BP.
XX
AC X75457; L26350;
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 YCplac111 - complete.
XX
KW cloning vector.
XX
OS Cloning vector
OC Artificial sequences; Cloning vehicles.
XX
RN [1]
RP 1-6112
RC YCplac111
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 1-6112
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
RC [pTC1 from pT322 & CEN4]
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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RN [3]
RC YRpmini7 from YRp17
RC from pNN279 & YRpmini7
RC from pNN280 series & YRpmini7
RA Mann C., Davis R.M.;
RT "Structure and sequence of the centromeric DNA of chromosome 4 in
RT Saccharomyces cerevisiae";
RL Mol. Cell. Biol. 6:241-245(1986).
XX
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: 415325
CC NM (YCplac111)
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)(YEplac112)(YEplac181)(YEplac195)(YIplac128)
CC BR (YIplac204)(YIplac211)
CC OF ()
CC OR ()
XX
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 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
FT 2. plasmid2 HpaI-AccI 1600bp, LEU2 gene
FT -> plasmid3 4500bp
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 HpaI-AccI 1600bp, LEU2 gene
FT 2. pAC5 EcoRI 6700bp
FT -> plasmid4 8300bp
FT 1. plasmid4 HindIII-HindIII 3700bp
FT mung bean exonuclease
FT AatII-AatII 3680bp
FT 2. pUC19 AatII-NdeI 2437bp 185..2622
FT :Klenow
FT -> YCplac111 6112bp"
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 6112 BP; 1756 A; 1314 C; 1291 G; 1751 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 attaattcta ccctatgaac atattccatt ttgtaatttc
gtgtcgtttc tattatgaat ttcatttata aagtttatgt acaaatatca taaaaaaaga
gaatcttttt aagcaaggat tttcttaact tcttcggcga cagcatcacc gacttcggtg
gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac ctttttaact
gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa catcattgca
gcagacaaga tagtggcgat agggtcaacc ttattctttg gcaaatctgg agcagaaccg
tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc caaggacgca
gatggcaaca aacccaagga acctgggata acggaggctt catcggagat gatatcacca
aacatgttgc tggtgattat aataccattt aggtgggttg ggttcttaac taggatcatg
gcggcagaat caatcaattg atgttgaacc ttcaatgtag gaaattcgtt cttgatggtt
tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt atccaaggac
caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct tgtgattctt
tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc atcgtcttcc
tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa gtcagtacct
ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc aaagttacat
ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc ttgttcaggt
ctaacactac ctgtacccca tttaggacca cccacagcac ctaacaaaac ggcatcaacc
ttcttggagg cttccagcgc ctcatctgga agtgggacac ctgtagcatc gatagcagca
ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc agaaatagct
ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc tggcaaaacg
acgatcttct taggggcaga cataggggca gacattagaa tggtatatcc ttgaaatata
tatatatatt gctgaaatgt aaaaggtaag aaaagttaga aagtaagacg attgctaacc
acctattgga aaaaacaata ggtccttaaa taatattgtc aacttcaagt attgtgatgc
aagcatttag tcatgaacgc ttctctattc tatatgaaaa gccggttccg gcctctcacc
tttccttttt ctcccaattt ttcagttgaa aaaggtatat gcgtcaggcg acctctgaaa
ttaacaaaaa atttccagtc atcgaatttg attctgtgcg atagcgcccc tgtgtgttct
cgttatgttg aggaaaaaaa taatggttgc taagagattc gaactcttgc atcttacgat
acctgagtat tcccacagtt aattcttgaa gacgaaaggg 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
//