PpUC18 Not vector
Ed. 1 © 2006 Biomedal, S.L.
Web: www.biomedal.com
.
Email: [email protected]
User's manual
Biomedal
NOTES
pUC18Not vector
For research use only
1. Product Description ..................................................................2
1.1. pUC18Not vector ......................................................................2
1.2. pUC18-pUC18Not vectors common features ..............................3
2. pUC18Not map
pUC18Not vector
Contents
.......................................................................4
3. Sequence and restriction analysis .........................................5
4. Multiple cloning site ...............................................................10
5. References .....................................................................................11
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1
1. Product Description.
5. References.
1.1. pUC18Not vector.
Selection for resistance to
Ampicillin
Replication Origin
pMB1 replicon rep (pBR322)
Antibiotic Resistance
Ampicillin
Host strain
Copy number
E. coli strains
High
2
1. Yanisch-Perron C., Vieira J. And Messing J. “Improved M13 phage
cloning vectors and host strain: nucleotide sequences of the M13mp18 and
pUC19 vectors. Gene 33, 103-119, 1985.
pUC18Not vector
pUC18Not is a 2715 basepair plasmid derived from pUC18 vector
(GenBank/EMBL sequence accession number L09136), so it can be used as
cloning vector in any E. coli strain and exhibit the blue/white screening property
in conjunction with X-Gal containing plates. pUC18Not carries pUC18 multiple
cloning site polylinker (in the same orientation as M13mp18), flanked by Not
I cleavage sequence. This vector is an excellent auxiliary plasmid to clone
fragments, that will be introduce as Not fragments in other vectors with this
restriction site.
11
PRODUCT
QUANTITY
STORAGE
CAT. NO
pUC18Not
8 mg
4ºC or -20ºC
CV-3282
! NOTE:
• The plasmid is shipped at room temperature. Upon arrival, store it according to the
direction in the table above.
•The plasmid is supplied dried.
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4. Multiple cloning site.
1.1. pUC18-pUC18Not vectors common features.
Because pUC18Not is a derivative vector from pUC18, both of vectors
have common features. These vectors contain:
M13 pUC (-47)
M13 pUC (-20)
GGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTGGGCGGCCGC
CCCATTGCGGTCCCAAAAGGGTCAGTGCTGCAACATTTTGCTGCCGGTCACGGTTCGACCCGCCGGCG
SmaI
CCCGGG
HindIII SphI PstI SalI XbaI BamHI
KpnI SacI EcoRI
NotI
AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCGCGCGGCCGCA
TTCGAACGTACGGACGTCCAGCTGAGATCTCCTAGGGGCCCATGGCTCGAGCTTAAGCGCGCCGGCGT
10
ATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACG
TAAGCATTAGTACAGTATCGACAAAGGACACACTTTAACAATAGGCGAGTGTTAAGGTGTGTTGTATGC
MET
b-galactosidase
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M13 pUC reverse (-48)
· pMB1 replicon rep, as origin of replication.
· bla gene, which confers resistance to ampicillin.
NotI · E. coli operon lac region, that contains CAP protein binding site,
promoter Plac, lac repressor binding site and 5’-terminal region of the
lacZ gene encoding the N-terminal fragment of b-galactosidase. This
fragment is capable of alpha-complementation with a defective form
of b-galactosidase encoding by host. The synthesis of b-galactosidase
N-terminal fragment is induced by IPTG. In presence of IPTG, both
fragments of the enzyme are synthesised and form blue colonies on
media with X-Gal. Insertion of DNA into the MCS abolishes the alpha
complementation. Bacteria carrying recombinant plasmids will give
rise to white colonies.
!
NOTE: pUC18Not carries a single mutation that eliminates a C and changes the
position of the start codon of lacZ gene (493-Met instead of 498-Met). This
change eliminates the first and second aminoacids of b-galactosidase
without changing the structure and function of the lacZ alpha product.
pUC18Not vector
M13 pUC (-40)
3
2. pUC18Not MAP.
RESTRICTION ANALYSIS
RESTRICTION ENZYMES THAT DO NOT CUT pUC18Not
lacZ a
bla
Not I (475)
Plac
pUC18Not
2715 bp
ori
4
Blp I
BmgB I
Bmt I
Bpl I
Bpu10 I
BsaA I
BsaB I
BseR I
Bsg I
BsiW I
Bsm I
BsmF I
BspE I
BsrG I
BssH II
BstB I
BstE II
BstX I
BstZ17 I
Bsu36 I
Btg I
Cla I
Dra III
EcoN I
EcoR V
Fal I
Fse I
FspA I
Hpa I
Mfe I
Mlu I
Msc I
Psr I
Rsr II
Sac II
SanD I
SexA I
Sfi I
Sgf I
SgrA I
SnaB I
Spe I
Srf I
Stu I
Sty I
Swa I
Tth111 I
Xho I
Nae I
Nco I
NgoMI V
Nhe I
Nru I
Nsi I
Oli I
Pac I
PflM I
Pme I
Pml I
Ppu10 I
PpuM I
PshA I
Psi I
Psp0M I
9
SEQUENCE REFERENCE POINT
Multiple Cloning Site: 406-480
lacZ: 149-396/486-493 (complementary strand)
lacZ start codon: 493 (complementary strand)
lacZ promoter: 499-572 (complementary strand)
ColE1 origin (origin pUC): 837-1510 (complementary strand)
bla coding region (ampicillin resistance): 1658-2515 (complementary
strand)
bla promoter: 2516-2614 (complementary strand)
Binding site of pUC/M13 Forward Sequencing Primer (-20): 379-395
Binding site of pUC/M13 Forward Sequencing Primer (-40): 359-375
Binding site of pUC/M13 Forward Sequencing Primer (-47): 352-375
Binding site of pUC/M13 Reverse Sequencing Primer (-48): 506-529
(complementary strand)
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pUC18Not vector
Not I (408)
MCS
Aar I
Afe I
Afl II
Age I
Ale I
Alo I
Apa I
Asc I
AsiS I
Avr II
Bae I
Bbs I
BbvC I
Bcl I
BfrB I
Bgl II
RESTRICTION ENZYMES THAT CUT ONCE pUC18Not
Aat II
Acc I
Acc65 I
Afl III
Ahd I
AlwN I
Apo I
Ava I
BamH I
Ban II
Bbe I
Bcg I
Bpm I
Bsa I
BsaX I
BspM I
BsrF I
BstAP I
Ecl136 II
Eco0109 I
EcoR I
Hinc II
HinD III
Kas I
Kpn I
Nar I
Nde I
Pci I
Pfo I
Pst I
Sac I
Sal I
Sap I
Sbf I
Sca I
! Underlined: Underlined multicloning restriction sites.
Sfo I
Sma I
Sph I
Ssp I
Xba I
Xcm I
Xma I
Xmn I
Zra I
TspGWI
2151 CTGCATAATT CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC
GACGTATTAA GAGAATGACA GTACGGTAGG CATTCTACGA AAAGACACTG
S C L
E R V T
M G D
T L H
K E T V
TatI
ScaI
BcgI
2201 TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG CGGCGACCGA
ACCACTCATG AGTTGGTTCA GTAAGACTCT TATCACATAC GCCGCTGGCT
AclI
XmnI
2301 ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC
TGAAATTTTC ACGAGTAGTA ACCTTTTGCA AGAAGCCCCG CTTTTGAGAG
PpiI
TaqII
2351 AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC
TTCCTAGAAT GGCGACAACT CTAGGTCAAG CTACATTGGG TGAGCACGTG
Eco57I
TaqII
2401 CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA
GGTTGACTAG AAGTCGTAGA AAATGAAAGT GGTCGCAAAG ACCCACTCGT
TspGWI
2451 AAAACAGGAA GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA
TTTTGTCCTT CCGTTTTACG GCGTTTTTTC CCTTATTCCC GCTGTGCCTT
8
SspI
2501 ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA AGCATTTATC
TACAACTTAT GAGTATGAGA AGGAAAAAGT TATAATAACT TCGTAAATAG
BciVI
2551 AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT
TCCCAATAAC AGAGTACTCG CCTATGTATA AACTTACATA AATCTTTTTA
ZraI
AatII
2601 AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT
TTTGTTTATC CCCAAGGCGC GTGTAAAGGG GCTTTTCACG GTGGACTGCA
2651 CTAAGAAACC ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTATCA
GATTCTTTGG TAATAATAGT ACTGTAATTG GATATTTTTA TCCGCATAGT
EcoO109I BsmBI
2701 CGAGGCCCTT TCGTC
GCTCCGGGAA AGCAG
PfoI
1 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG
AGCGCGCAAA GCCACTACTG CCACTTTTGG AGACTGTGTA CGTCGAGGGC
BsmBI
DrdI
51 GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG
CTCTGCCAGT GTCGAACAGA CATTCGCCTA CGGCCCTCGT CTGTTCGGGC
101 TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG
AGTCCCGCGC AGTCGCCCAC AACCGCCCAC AGCCCCGACC GAATTGATAC
NdeI
TatI
BstAPI
151 CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA
GCCGTAGTCT CGTCTAACAT GACTCTCACG TGGTATACGC CACACTTTAT
SfoI
NarI
KasI
BbeI
BglI
201 CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT
GGCGTGTCTA CGCATTCCTC TTTTATGGCG TAGTCCGCGG TAAGCGGTAA
FspI
PvuI
251 CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT
GTCCGACGCG TTGACAACCC TTCCCGCTAG CCACGCCCGG AGAAGCGATA
PvuII
301 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA
ATGCGGTCGA CCGCTTTCCC CCTACACGAC GTTCCGCTAA TTCAACCCAT
XcmI
BmrI
BceAI
351 ACGCCAGGGT TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGCCAA
TGCGGTCCCA AAAGGGTCAG TGCTGCAACA TTTTGCTGCC GGTCACGGTT
SalI
XmaI
BspMI
SmaI
NotI
SphI
PstI
HincII
AvaI
BseYI EagI
HindIII
SbfI
AccI XbaI
BamHI
401 GCTGGGCGGC CGCAAGCTTG CATGCCTGCA GGTCGACTCT AGAGGATCCC
CGACCCGCCG GCGTTCGAAC GTACGGACGT CCAGCTGAGA TCTCCTAGGG
SacI
EcoICRI
KpnI BanII EcoRI
EagI
Acc65I
ApoI
NotI
451 CGGGTACCGA GCTCGAATTC GCGCGGCCGC AATTCGTAAT CATGTCATAG
GCCCATGGCT CGAGCTTAAG CGCGCCGGCG TTAAGCATTA GTACAGTATC
501 CTGTTTCCTG TGTGAAATTG TTATCCGCTC ACAATTCCAC ACAACATACG
GACAAAGGAC ACACTTTAAC AATAGGCGAG TGTTAAGGTG TGTTGTATGC
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pUC18Not vector
2251 GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA
CAACGAGAAC GGGCCGCAGT TATGCCCTAT TATGGCGCGG TGTATCGTCT
3. Sequence and restriction analysis.
5
551 AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTAAC
TCGGCCTTCG TATTTCACAT TTCGGACCCC ACGGATTACT CACTCGATTG
601 TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG
AGTGTAATTA ACGCAACGCG AGTGACGGGC GAAAGGTCAG CCCTTTGGAC
PvuII
TfiI
BsaXI
651 TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT
AGCACGGTCG ACGTAATTAC TTAGCCGGTT GCGCGCCCCT CTCCGCCAAA
1401 TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA
ACTAGGCCGT TTGTTTGGTG GCGACCATCG CCACCAAAAA AACAAACGTT
1451 GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT
CGTCGTCTAA TGCGCGTCTT TTTTTCCTAG AGTTCTTCTA GGAAACTAGA
PpiI
1501 TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT
AAAGATGCCC CAGACTGCGA GTCACCTTGC TTTTGAGTGC AATTCCCTAA
751 TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG
AGCAAGCCGA CGCCGCTCGC CATAGTCGAG TGAGTTTCCG CCATTATGCC
1551 TTGGTCATGA GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA
AACCAGTACT CTAATAGTTT TTCCTAGAAG TGGATCTAGG AAAATTTAAT
PciI
TfiI
AflIII
801 TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG
AATAGGTGTC TTAGTCCCCT ATTGCGTCCT TTCTTGTACA CTCGTTTTCC
1601 AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA ACTTGGTCTG
TTTTACTTCA AAATTTAGTT AGATTTCATA TATACTCATT TGAACCAGAC
DrdI
901 ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG
TATCCGAGGC GGGGGGACTG CTCGTAGTGT TTTTAGCTGC GAGTTCAGTC
951 AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG
TCCACCGCTT TGGGCTGTCC TGATATTTCT ATGGTCCGCA AAGGGGGACC
MmeI
BciVI
1001 AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC
TTCGAGGGAG CACGCGAGAG GACAAGGCTG GGACGGCGAA TGGCCTATGG
1651 ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA
TGTCAATGGT TACGAATTAG TCACTCCGTG GATAGAGTCG CTAGACAGAT
AhdI
1701 TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT
AAAGCAAGTA GGTATCAACG GACTGAGGGG CAGCACATCT ATTGATGCTA
BmrI
BsrDI
BsaI
1751 ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC
TGCCCTCCCG AATGGTAGAC CGGGGTCACG ACGTTACTAT GGCGCTCTGG
BsrFI BpmI
BglI
1801 CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG
GTGCGAGTGG CCGAGGTCTA AATAGTCGTT ATTTGGTCGG TCGGCCTTCC
1051 TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC
ACAGGCGGAA AGAGGGAAGC CCTTCGCACC GCGAAAGAGT ATCGAGTGCG
AvaII
1851 GCCGAGCGCA GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT
CGGCTCGCGT CTTCACCAGG ACGTTGAAAT AGGCGGAGGT AGGTCAGATA
BseYI
1101 TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT
ACATCCATAG AGTCAAGCCA CATCCAGCAA GCGAGGTTCG ACCCGACACA
FspI
1901 TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT AATAGTTTGC
ATTAACAACG GCCCTTCGAT CTCATTCATC AAGCGGTCAA TTATCAAACG
1151 GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC
CGTGCTTGGG GGGCAAGTCG GGCTGGCGAC GCGGAATAGG CCATTGATAG
AclI
BsrDI
1951 GCAACGTTGT TGCCATTGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT
CGTTGCAACA ACGGTAACGA TGTCCGTAGC ACCACAGTGC GAGCAGCAAA
MmeI
AlwNI
1201 GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC
CAGAACTCAG GTTGGGCCAT TCTGTGCTGA ATAGCGGTGA CCGTCGTCGG
1251 ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT
TGACCATTGT CCTAATCGTC TCGCTCCATA CATCCGCCAC GATGTCTCAA
BceAI
1301 CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGGACA GTATTTGGTA
GAACTTCACC ACCGGATTGA TGCCGATGTG ATCTTCCTGT CATAAACCAT
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pUC18Not vector
SapI
701 GCGTATTGGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG
CGCATAACCC GCGAGAAGGC GAAGGAGCGA GTGACTGAGC GACGCGAGCC
851 CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC
GGTCGTTTTC CGGTCCTTGG CATTTTTCCG GCGCAACGAC CGCAAAAAGG
6
Eco57I
1351 TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT
AGACGCGAGA CGACTTCGGT CAATGGAAGC CTTTTTCTCA ACCATCGAGA
2001 GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG
CCATACCGAA GTAAGTCGAG GCCAAGGGTT GCTAGTTCCG CTCAATGTAC
AvaII
PvuI
2051 ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG
TAGGGGGTAC AACACGTTTT TTCGCCAATC GAGGAAGCCA GGAGGCTAGC
2101 TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA
AACAGTCTTC ATTCAACCGG CGTCACAATA GTGAGTACCA ATACCGTCGT
7