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ID YCPLAC33 preliminary; circular DNA; SYN; 5603 BP.
XX
AC X75456; L26352;
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 YCplac33 - complete.
XX
KW cloning vector.
XX
OS Cloning vector
OC Artificial sequences; Cloning vehicles.
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RN [1]
RP 1-5603
RC YCplac33
RA Gietz R.D.;
RT ;
RL Submitted (13-MAY-1993) by:
RL Gietz R.D., Health Sciuence Ctr., University of Manitoba,
RL Manitoba, CANADA.
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RN [2]
RP 1-5603
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: 415329
CC NM (YCplac33)
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)(YCplac111)(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 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. 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 HindIII-SmaI 1108bp 2273..3381, URA3 gene
FT 2. pAC5 EcoRI 6700bp
FT -> plasmid4 7800bp
FT 1. plasmid4 HindIII-HindIII 3200bp
FT mung bean exonuclease
FT AatII-AatII 3200bp
FT 2. pUC19 AatII-NdeI 2437bp 185..2622
FT :Klenow
FT -> YCplac111 5603bp"
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)"
XX
SQ Sequence 5603 BP; 1586 A; 1232 C; 1156 G; 1629 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 attgggtaat aactgatata attaaattga agctctaatt
tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat
cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc
cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca
tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg
ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata
aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct
ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc
tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg
tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat
ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg
gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc
acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc
ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta
aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc
gacatgattt atcttcgttt cctgcatgtt tttgttctgt gcagttgggt taagaatact
gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc
tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaagaaac
cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga attgaaaagc taattcttga
agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
tcagtgagcg aggaagcgga aga
//