7/27/2019 pUC18-pUC19-map.pdf

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Reerence

1. Yanisch-Perron, C., et al., Improved M13 phage cloning

vectors and host strains: nucleotide sequences o the

M13mp18 and pUC19 vectors, Gene, 33, 103-119, 1985.

pUC18, pUC19

Enzymes which cut pUC18 DNA once:

AatII 2617Acc65I* 438

 AIII 806BamHI* 429BcgI 2215BsaXI 659BstAPI 179BveI* 413CaiI 1217Cr9I* 434Cr10I 1779Eam1105I 1694Ecl136II* 444Eco24I* 444Eco31I 1766Eco88I* 434EcoO109I 2674EcoRI* 450

EheI 235GsuI 1784HincII* 417

HindIII* 399KpnI* 438LguI 683NdeI 183NmeAIII 1822PaeI* 405PdmI 2294PoI 46PscI 806PspFI 1110PstI* 411SacI* 444SalI* 417ScaI 2177SdaI* 410SmaI* 434SspI 2501SspDI 235

XapI* 450XbaI* 423XmiI* 417

* MCS

pUC18 and pUC19 vectors are small, high copy

number, E.coli plasmids, 2686 bp in length. They are

identical except that they contain multiple cloning sites

(MCS) arranged in opposite orientations. pUC18/19

plasmids contain: (1) the pMB1 replicon rep responsibleor the replication o the plasmid (source – plasmid

pBR322). The high copy number o pUC plasmids is

a result o the lack o the rop gene and a single point

mutation in the replicon rep o pMB1; (2) the bla  

gene, coding or β-lactamase, that coners resistance

to ampicillin (source – plasmid pBR322). It diers

rom that o pBR322 by two point mutations; (3) the

region o E.coli  lac operon containing a CAP protein

binding site, promoter Plac, lac repressor binding site

and the 5’-terminal part o the lacZ gene encoding

the N-terminal ragment o β-galactosidase (source –

M13mp18/19). This ragment, whose synthesis

can be induced by IPTG, is capable o intra-allelic

( α ) complementation with a deective orm o

β-galactosidase encoded by the host (mutation ∆(lacZ) 

M15  ). In the presence o IPTG, bacteria synthesize

both ragments o the enzyme and orm blue colonies

on media with X-Gal. Insertion o DNA into the MCSlocated within the lacZ gene (codons 6-7 o lacZ are

replaced by MCS) inactivates the N-terminal ragment

o β-galactosidase and abolishes α-complementation.

Bacteria carrying recombinant plasmids thereore give

rise to white colonies.

The map shows enzymes that cut pUC18/19 DNA

once. Thermo Scientifc enzymes are shown in orange.

The coordinates reer to the position o the frst

nucleotide in each recognition sequence.

The exact positions o the genetic elements are shown

on the map (termination codons included). The bla  

gene nucleotides 2486-2418 (complementary strand)

code or a signal peptide. The LacZ polypeptide

corresponding to wt β-galactosidase and essential or

blue/white screening ends at nt position 236 (compl.

strand); another 30 codons in the same reading rame

are derived rom pBR322. The indicated rep region

is sufcient to promote replication. DNA replicationinitiates at position 866 (+/- 1) and proceeds in the

direction indicated. Plasmids carrying the pMB1 and

ColE1 replicons are incompatible, but they are ully

compatible with those carrying the p15A replicon

(pACYC177, pACYC184). pMB1-derived plasmids can

be amplifed using chloramphenicol.

GenBank/EMBL Accession Numbers

For pUC18 – L09136;

or pUC19 – L09137.

Additional Inormation• CAPproteinbindingsite–591-554(compl.strand);

• mRNA(LacZ)startsatntposition507(compl.strand);

• lac repressor binding site – 507-487 (compl. strand).

Multiple Cloning Site o pUC18

M13/pUC sequencing primer, 17-mer (-20), (#S0100) PstI  HincII  Cr9I Ecl136II

SdaI SalI Eco88I Acc65I Eco24I EcoRI

399 HindIII PaeI BveI XmiI XbaI BamHI  SmaI KpnI SacI XapI 455

5’ - G TAA AAC GAC GGC CAG TGC C AA GCT TGC ATG CCT GCA GGT CGA CTC TAG AGG ATC CCC GGG TAC CGA GCT CGA ATT CGT AAT CAT GGT CAT AGC TGT TTC CTG - 3’

3’ - C ATT TTG CTG CCG GTC A CG GTT CGA ACG TAC GGA CGT CCA GCT GAG ATC TCC TAG GGG CCC ATG GCT CGA GCT TAA GCA TTA GTA CCA  GTA  TCG ACA AAG GAC - 5’

  Val Val Ala Leu Ala Leu Ser Ala His Arg Cys Thr Ser Glu Leu Pro Asp Gly Pro Val Ser Ser Ser Asn Thr Ile Met Thr Met 

M13/pUC reverce sequencing primer, 17-mer (-26), (#S0101)

Multiple Cloning Site o pUC19

M13/pUC sequencing primer, 17-mer (-20), (#S0100) Ecl136II  Cr9I  HincII  PstI 

EcoRI  Eco24I  Acc65I  Eco88I  SalI BveI

396 XapI SacI KpnI SmaI BamHI  XbaI XmiI SdaI PaeI HindIII 452

5’ - G TAA AAC GAC GGC CAG TGA ATT CGA GCT CGG TAC CCG GGG ATC CTC TAG AGT CGA CCT GCA GGC ATG CAA GCT TGG CGT AAT CAT GGT CAT AGC TGT TTC CTG - 3’

3’ - C ATT TTG CTG CCG GTC  A CT TAA GCT CGA GCC ATG GGC CCC TAG GAG ATC TCA GCT GGA CGT CCG TAC GTT CGA A CC CGA TTA GTA CCA  GTA  TCG ACA AAG GAC - 5’

  Val Val Ala Leu Ala Leu Ser Ala His Arg Cys Thr Ser Glu Leu Pro Asp Gly Pro Val Ser Ser Ser Asn Thr Ile Met Thr Met 

M13/pUC reverce sequencing primer, 17-mer (-26), (#S0101)

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There are no restriction sites in pUC18 DNA or the ollowing enzymes:

AanI, AarI, AbsI, AdeI, AjiI, AjuI, AlI, AloI, ApaI, BaeI, BbvCI, BclI, BcuI,

BglII, BoxI, BpiI, BplI, Bpu10I, Bpu1102I, BseJI, BseRI, BsgI, BshTI, Bsp68I,Bsp119I, Bsp120I, Bsp1407I, BspOI, BspTI, Bst1107I, BstXI, Bsu15I, BtgI,

BtgZI, Cr42I, CpoI, CspCI, Eco32I, Eco47III, Eco52I, Eco72I, Eco81I, Eco91I,

Eco105I, Eco130I, Eco147I, FalI, FaqI, FseI, FspAI, Kpn2I, KspAI, MlsI, MluI,

Mph1103I, MreI, MssI, MunI, Mva1269I, NcoI, NheI, NotI, OliI, PacI, PasI,

PauI, PdiI, P23II, Ppu21I, Psp5II, PspXI, PsrI, PsyI, SanDI, SexAI, SaAI, SfI,

SgI, SgrAI, SgrDI, SgsI, SmiI, SrI, Van91I, XagI, XcmI, XhoI, XmaJI.

Indicates restriction sites o Thermo Scientifc enzymes that are sensitive (cleavage completely

blocked or partially inhibited) to overlapping Dam/Dcm methylation. Complete cleavage is achieved with DNA

substrates isolated rom dam – or dcm – hosts.

EnzymeNumber o recognition sites

1 2 3 4 5 6 7 8 9 10 11 12

DrdI 91 908

AatII 2617

Acc65I 438

 AIII 806

Alw21I 177 444 1120 2281 2366

Alw26I 51 1766 2531 2684

Alw44I 177 1120 2366BamHI 429

BauI 979 2363 2670

BccI 1735 1859 2146

BceAI 387 1292

BcgI 2215

BcnI 47 82 434 435 1185 1881 2232

BmrI 364 1744

BmI 411 1071 1262 1940

BuI 1015 2542

BglI 245 1813

Bme1390I 47 82 354 434 435 545 833 954 967 1185 1881 2232

BpuEI 912 1174 1451 2319

BsaWI 1012 1159 1990

BsaXI 659

BseDI 354 433 434 545 966

BseGI 77 321 1679 1860 2147

BseLI 47 648 822 840 1006 1285

BseMI 1753 1935

BseMII 171 1081 1490 1656 2196

BseNI 365 391 606 1209 1222 1339 1745 1863 1906 2170 2345

BseSI 177 1120 2366BseXI 41 254 327 630 711 729 1148 1213 1216 1422 1750 2116

Bsh1236I 2 4 107 652 654 852 1433 1763 2256 2588

Bsh1285I 276 719 1143 2066 2215

BshNI 235 438 550 1647

BspLI 235 429 438 550 836 875 1647 1741 1782 1993 2583

BspPI 429 430 1373 1447 1459 1544 1557 2021 2324 2342

BstAPI 179

BsuRI 287 389 646 820 831 849 1283 1741 1821 2088 2675

BtsI 593 2092 2120

BveI 413

CaiI 1217

EaeI 388 645 2087

Cr9I 434

Cr10I 1779

Cr13I 286 1741 1820 1837 2059 2675

CseI 108 908 1486 2236

Csp6I 168 439 2178

DraI 1563 1582 2274

Eam1104I 290 684 2488

Eam1105I 1694

EciI 878 1024 1852

Ecl136II 444

Eco24I 444

Eco31I 1766

Eco47I 1837 2059

Eco57I 1333 2381

Eco88I 434

EcoO109I 2674

EcoP15I 1214 1330 1423

EcoRI 450

EcoRII 354 545 833 954 967

EheI 235

Esp3I 51 2683

FokI 77 321 1679 1860 2147

FspBI 424 1301 1554 1889

GsuI 1784

HaeII 235 680 1050

EnzymeNumber o recognition sites

1 2 3 4 5 6 7 8 9 10 11 12

Hin1I 235 2235 2617

Hin1II 38 406 461 807 1527 2018 2028 2106 2142 2535 2640

HincII 417

HindIII 399

HinI 420 641 706 781 1177 1694

HphI 12 21 1550 1777 2173 2399 2414

Hpy8I 177 417 1120 1608 2366Hpy99I 372 385 907 1701 1964

Hpy188I 31 156 918 996 1349 1483 1618 2064 2075 2195

HpyAV 270 1036 1304 1346 1817 2054 2429

HpyF3I 171 1081 1490 1656 2196 2622

KpnI 438

LguI 683

Lsp1109I 41 254 327 630 711 729 1148 1213 1216 1422 1750 2116

LweI 78 153 208 229 894 1946 2156 2386

MaeIII 57 348 368 1163 1226 1342 1625 1956 2014 2167 2355

MbiI 496 737 2538

MboII 291 685 1456 1547 2302 2380 2489

MmeI 996 1180

MspA1I 112 306 628 1146 1391 2332

MvaI 354 545 833 954 967

NdeI 183

NmeAIII 1822

NmuCI 57 368 1956 2167

NsbI 256 1919

PaeI 405

PagI 1526 2534 2639

PdmI 2294PeI 641 781

PoI 46

PscI 806

Psp1406I 1924 2297

PspFI 1110

PstI 411

PsuI 429 1447 1458 1544 1556 2324 2341

PvuI 276 2066

PvuII 306 628

RsaI 168 439 2178

RseI 1947 2106 2465

SacI 444

SalI 417

ScaI 2177

SchI 420 706 1177 1694

SdaI 410

SduI 177 444 1120 2281 2366

SmaI 434

SmoI 912 1174 1451 2319

FauI  114 124 284 598 655

SspI 2501

SspDI 235

TaaI 19 54 262 768 839 1309 1622 2137

TaiI 374 1509 1925 2298 2618

TaqI 418 448 906 2350

TasI 451 487 504 579 1567 1873 2128

TatI 167 2177

TauI 150 732 850 1005 2089 2211 2440

TscAI 392 593 702 1208 1221 1492 1641 1746 2093 2120

TseI 41 254 327 630 711 729 1148 1213 1216 1422 1750 2116

TspDTI 639 1575 1677 1980

TspGWI 2149 2466

VspI 576 635 1870

XapI 450

XbaI 423

XceI 37 405 806

XmiI 417

pUC18 DNA Restriction Sites