puc18-puc19-map

puc18-puc19-map pUC18 and pUC19 vectors are small, high copy ( ) complementation with a defective form of corresponding to wt -galactosidase and essential for number, E.coli plasmids, 2686 bp in length. They are -galactosidase encoded by the host (mutation (lacZ) blue/white screening ends at nt position 236 (compl. identical except that they contain multiple cloning sites M15). In the presence of IPTG, bacteria synthesize strand); another 30 codons in the same reading frame (MCS) arranged in opposite orientations. pUC18/19 both fragments of the enzyme and form blue colonies are derived from pBR322. The indicated rep region plasmids contain: (1) the pMB1 replicon rep responsible ,cient to promote replication. DNA replication on media with X-Gal. Insertion of DNA into the MCS is suffor the replication of the plasmid (source – plasmid initiates at position 866 (+/- 1) and proceeds in the located within the lacZ gene (codons 6-7 of lacZ are pBR322). The high copy number of pUC plasmids is direction indicated. Plasmids carrying the pMB1 and replaced by MCS) inactivates the N-terminal fragment a result of the lack of the rop gene and a single point ColE1 replicons are incompatible, but they are fully of -galactosidase and abolishes -complementation. mutation in the replicon rep of pMB1; (2) the bla compatible with those carrying the p15A replicon Bacteria carrying recombinant plasmids therefore give gene, coding for -lactamase, that confers resistance (pACYC177, pACYC184). pMB1-derived plasmids can rise to white colonies. to ampicillin (source – plasmid pBR322). It differs be ampli,ed using chloramphenicol. The map shows enzymes that cut pUC18/19 DNA from that of pBR322 by two point mutations; (3) the ,c enzymes are shown in orange. once. Thermo ScientiGenBank/EMBL Accession Numbers region of E.coli lac operon containing a CAP protein The coordinates refer to the position of the ,rst For pUC18 – L09136; binding site, promoter Plac, lac repressor binding site nucleotide in each recognition sequence. for pUC19 – L09137. and the 5’-terminal part of the lacZ gene encoding The exact positions of the genetic elements are shown the N-terminal fragment of -galactosidase (source – on the map (termination codons included). The bla Additional Information M13mp18/19). This fragment, whose synthesis gene nucleotides 2486-2418 (complementary strand) • CAP protein binding sitecan be induced by IPTG, is capable of intra-allelic code for a signal peptide. The LacZ polypeptide – 591-554 (compl. strand); • mRNA (LacZ) starts at nt position 507 (compl. strand); Enzymes which cut pUC18 DNA once: • lac repressor binding site – 507-487 (compl. AatII 2617 HindIII* 399 strand). Acc65I* 438 438 KpnI* A,III 806 LguI 683 BamHI* 429 NdeI 183 BcgI 2215 NmeAIII 1822 BsaXI 659 PaeI* 405 179 BstAPI PdmI 2294 413 BveI* PfoI 46 PscI 1217 CaiI 806 Cfr9I* PspFI 434 1110 Cfr10I PstI* 1779 411 SacI* Eam1105I 1694 444 Ecl136II* SalI* 444 417 ScaI Eco24I* 444 2177 SdaI* Eco31I 1766 410 SmaI* 434 Eco88I* 434 SspI 2674 EcoO109I 2501 SspDI 450 EcoRI* 235 XapI* 235 450 GsuIEheI XbaI* 1784 423 HincII* XmiI* 417 417 * MCS Multiple Cloning Site of pUC18 M13/pUC sequencing primer, 17-mer (-20), (#S0100) PstI HincII Cfr9I 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 CAA 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 ACG 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 of pUC19 M13/pUC sequencing primer, 17-mer (-20), (#S0100) Ecl136II Cfr9I 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 ACT TAA GCT CGA GCC ATG GGC CCC TAG GAG ATC TCA GCT GGA CGT CCG TAC GTT CGA ACC 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) Reference 1. Yanisch-Perron, C., et al., Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors, Gene, 33, 103-119, 1985. pUC18 DNA Restriction Sites DrdI 91 908 Hin1I 235 2235 2617 406 461 AatII 2617 Hin1II 38 807 1527 2018 2028 2106 2142 2535 2640 Acc65I 438 HincII 417 A,III 806 HindIII 399 Alw21I 420 177 444 1120 2281 2366 HinfI 641 706 781 1177 1694 Alw26I 51 1766 2531 2684 HphI 12 21 1550 1777 2173 2399 2414 1120 2366 Alw44I 177 Hpy8I 177 1120 1608 2366 417 BamHI 429 Hpy99I 372 385 907 1701 1964 BauI 979 2363 2670 Hpy188I 31 156 918 996 1349 1483 1618 2064 2075 2195 2146 2429 BccI 1859 270 1036 1304 1817 2054 1735 HpyAV 1346 BceAI 387 1292 HpyF3I 171 1081 1490 1656 2196 2622 BcgI 2215 KpnI 438 BcnI 47 82 434 435 1185 1881 2232 LguI 683 BmrI 364 1744 Lsp1109I 41 254 327 630 711 729 1148 1213 1216 1422 1750 2116 BfmI 1071 153 208 229 894 1946 2156 2386 411 1262 1940 LweI 78 2014 2167 2355 BfuI 1015 2542 MaeIII 57 348 368 1163 1226 1342 1625 1956 BglI 245 1813 MbiI 737 2538 496 435 545 833 Bme1390I 47 82 354 434 954 967 1185 1881 2232 MboII 291 685 1456 1547 2302 2380 2489 BpuEI 912 1174 1451 2319 MmeI 996 1180 2332 1159 1990 BsaWI 112 306 1012 MspA1I 628 1146 1391 BsaXI 659 MvaI 354 545 833 954 967 BseDI 354 433 434 545 966 NdeI 183 BseGI 77 321 1679 1860 2147 NmeAIII 1822 BseLI 47 648 822 840 1006 1285 NmuCI 57 368 1956 2167 1919 BseMI 1935 256 1753 NsbI BseMII 171 1081 1490 1656 2196 PaeI 405 1339 2639 BseNI 365 391 606 1209 1222 1745 1863 1906 2170 2345 PagI 1526 2534 2366 BseSI 177 1120 PdmI 2294 1750 781 BseXI 41 254 327 630 711 729 1148 1213 1216 1422 2116 PfeI 641 852 1433 1763 2256 2588 Bsh1236I 4 107 652 654 46 2 PfoI Bsh1285I 276 719 1143 2066 2215 PscI 806 BshNI 235 438 550 1647 Psp1406I 1924 2297 550 235 BspLI 429 438 836 875 1647 1741 1782 1993 2583 PspFI 1110 BspPI 429 430 1373 1447 1459 1544 1557 2021 2324 2342 PstI 411 BstAPI 179 429 PsuI 1447 1458 1544 1556 2324 2341 2675 BsuRI 287 389 646 820 831 849 1283 1741 1821 2088 PvuI 276 2066 BtsI 593 2092 2120 PvuII 306 628 413 439 BveI RsaI 168 2178 CaiI 1217 RseI 1947 2106 2465 EaeI 388 444 645 2087 SacI Cfr9I 434 SalI 417 Cfr10I 1779 ScaI 2177 286 SchI Cfr13I 1741 1820 1837 2059 2675 420 706 1177 1694 CseI 108 908 1486 2236 SdaI 410 444 1120 2281 Csp6I 168 439 2178 SduI 177 2366 DraI 1563 1582 2274 SmaI 434 Eam1104I 290 684 2488 SmoI 912 1174 1451 2319 124 284 598 1694 FauI 655 Eam1105I 114 EciI 878 1024 1852 SspI 2501 Ecl136II 444 SspDI 235 Eco24I 444 TaaI 19 54 262 768 839 1309 1622 2137 Eco31I 1766 TaiI 374 1509 1925 2298 2618 906 Eco47I 1837 TaqI 418 448 2350 2059 1873 2128 Eco57I 1333 2381 TasI 451 487 504 579 1567 Eco88I 434 TatI 167 2177 150 732 EcoO109I 2674 TauI 850 1005 2089 2211 2440 EcoP15I 1214 1330 1423 TscAI 392 593 702 1208 1221 1492 1641 1746 2093 2120 327 630 711 729 1148 1213 1216 1422 EcoRI 450 TseI 41 254 1750 2116 545 833 EcoRII 354 954 967 TspDTI 639 1575 1677 1980 EheI 235 TspGWI 2149 2466 635 Esp3I 576 51 2683 VspI 1870 FokI 77 321 1679 1860 2147 XapI 450 1301 1554 1889 FspBI XbaI 423 424 806 GsuI 1784 XceI 37 405 XmiI HaeII 235 680 1050 417 Indicates restriction sites of Thermo Scienti,c enzymes that are sensitive (cleavage completely There are no restriction sites in pUC18 DNA for the following enzymes: blocked or partially inhibited) to overlapping Dam/Dcm methylation. Complete cleavage is achieved with DNA AanI, AarI, AbsI, AdeI, AjiI, AjuI, AlfI, AloI, ApaI, BaeI, BbvCI, BclI, BcuI, – – substrates isolated from dam or dcm hosts. BglII, BoxI, BpiI, BplI, Bpu10I, Bpu1102I, BseJI, BseRI, BsgI, BshTI, Bsp68I, Bsp119I, Bsp120I, Bsp1407I, BspOI, BspTI, Bst1107I, BstXI, Bsu15I, BtgI, BtgZI, Cfr42I, 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, P,23II, Ppu21I, Psp5II, PspXI, PsrI, PsyI, SanDI, SexAI, SfaAI, S,I, SgfI, SgrAI, SgrDI, SgsI, SmiI, SrfI, Van91I, XagI, XcmI, XhoI, XmaJI.