Plasmid name: pUHG 16-3 Size: 7169 bp Resistance: Ampicillin Constructor: Manfred Gossen Host strain: XL1-blue, DH5 alpha, E.coli "SURE" Reference: Manfred Gossen unpublished
The plasmid consists out of four main fragments:
The plasmid was constructed by inserting a 3.4 kb XbaI fragment containing a modified lacZ gene (obtained from A. Leutz, MDC Berlin) into the XbaI- linearized vector pUHG10-3 (M.Gossen). The orientation of the insert was verified by sequencing. This plasmid is fully functional with respect to Tc-dependent ß-galactosidase expression in a tTA + background in stable as well as in transient experiments (M. Gossen, unpublished observation)
Afl II c/ttaag Fse I ggccgg/cc Pst I ctgca/g Age I a/ccggt Hind III a/agctt Rsr II cg/gwccg Avr II c/ctagg Kas I g/gcgcc Sal I g/tcgac Bbe I ggcgc/c Nae I gcc/ggc Sap I gcttcttc 1/4 BsaA I yac/gtr Nar I gg/cgcc Sfi I ggccnnnn/nggcc Bsm I gaatgc 1/-1 Nhe I g/ctagc SgrA I cr/ccggyg BspE I t/ccgga Not I gc/ggccgc SnaB I tac/gta BstB I tt/cgaa Nru I tcg/cga Spe I a/ctagt BstE II g/gtnacc Nsi I atgca/t Sph I gcatg/c Eag I c/ggccg Pac I ttaat/taa Sse8337 I cctgca/gg EcoN I cctnn/nnnagg Pml I cac/gtg Swa I attt/aaat Ehe I ggc/gcc PshA I gacnn/nngtc Tth111 I gacn/nngtc
Enzyme Position PaeR7 I c/tcgag 1 Xho I c/tcgag 1 Stu I agg/cct 336 Sac II ccgc/gg 439 Cla I at/cgat 1435 EcoR V gat/atc 1723 Dra III cacnnn/gtg 1797 BsaB I gatnn/nnatc 1939 Bcl I t/gatca 1958 BssH II g/cgcgc 2110 Eco47 III agc/gct 2445 Acc I gt/mkac 3375 Sna I gta/tac 3375 Xca I gta/tac 3375 Spl I c/gtacg 3383 Esp I gc/tnagc 3624 PpuM I rg/gwccy 3969 Nco I c/catgg 4076 Sty I c/cwwgg 4076 Apa I gggcc/c 4487 Bsp120 I g/ggccc 4487 Msc I tgg/cca 4630 Bgl II a/gatct 4661 Bsa I ggtctc 1/5 6245
Enzyme Sites Positions: Aat II gacgt/c 2 1231 7096 Ase I at/taat 2 5114 6349 Asp718 g/gtacc 2 304 461 BamH I g/gatcc 2 470 482 BspM I acctgc 4/8 2 1395 3267 Bsu36 I cc/tnagg 2 835 4590 Drd I gacnnnn/nngtc 2 2715 5387 EcoR I g/aattc 2 449 4581 Fsp I tgc/gca 2 752 6398 Hpa I gtt/aac 2 1036 1660 Kpn I ggtac/c 2 304 461 Sca I agt/act 2 4238 6656 Xba I t/ctaga 2 476 3940 Xcm I ccannnnn/nnnntgg 2 2822 3567 Xmn I gaann/nnttc 2 3695 6773 BstX I ccannnnn/ntgg 3 2825 3442 4633 EcoO109 I rg/gnccy 3 3969 4487 7153 Mlu I a/cgcgt 3 1914 2694 3119 Nsp I rcatg/y 3 3371 5031 5285 Nsp7524 I r/catgy 3 3371 5031 5285 NspC I rcatg/y 3 3371 5031 5285 PflM I ccannnn/ntgg 3 1851 2217 4861 Sma I ccc/ggg 3 308 465 487 Xma I c/ccggg 3 308 465 487
Aat II 2 BssH II 1 Hga I 13 Pml I - Acc I 1 BstB I - HgiA I 9 PpuM I 1 Afl II - BstE II - Hha I 41 PshA I - Afl III 6 BstK I 31 Hinc II 4 Pst I - Age I - BstN I 15 Hind III - Pvu I 4 Aha II 6 BstU I 37 Hinf I 13 Pvu II 4 Alu I 22 BstX I 3 HinP I 41 Rma I 6 Alw I 21 BstY I 10 Hpa I 2 Rsa I 11 AlwN I 5 Bsu36 I 2 Hpa II 31 Rsr II - Apa I 1 Cfr10 I 5 Hph I 15 Sac I 4 ApaL I 4 Cla I 1 Kas I - Sac II 1 Ase I 2 Csp6 I 11 Kpn I 2 Sal I - Asp718 2 Dde I 13 Mae II 13 Sap I - Ava I 6 Dpn I 33 Mae III 28 Sau3A I 33 Ava II 6 Dpn II 33 Mbo I 33 Sau96 I 16 Avr II - Dra I 6 Mbo II 17 Sca I 2 BamH I 2 Dra III 1 Mcr I 13 ScrF I 31 Ban I 7 Drd I 2 Mlu I 3 SfaN I 14 Ban II 6 Dsa I 7 Mme I 5 Sfe I 6 Bbe I - Dsa V 31 Mnl I 40 Sfi I - Bbs I 4 Eae I 5 Msc I 1 SgrA I - Bbv I 26 Eag I - Mse I 24 Sma I 3 BceF I 16 Ear I 4 Msp I 31 Sna I 1 Bcl I 1 Ecl136 I 4 Nae I - SnaB I - Bcn I 16 Eco47 III 1 Nar I - Spe I - Bgl I 4 Eco57 I 5 Nci I 16 Sph I - Bgl II 1 EcoN I - Nco I 1 Spl I 1 Bsa I 1 EcoO109 I 3 Nde I 5 Sse8337 I - BsaA I - EcoR I 2 Nhe I - Ssp I 4 BsaB I 1 EcoR II 15 Nla III 26 Stu I 1 BsaJ I 17 EcoR V 1 Nla IV 25 Sty I 1 Bsg I 7 Ehe I - Not I - Swa I - BsiE I 13 Esp I 1 Nru I - Taq I 23 BsiY I 16 Fau I 10 Nsi I - Tfi I 7 Bsm I - Fse I - Nsp I 3 Tth111 I - BsmA I 8 Fnu4H I 49 Nsp7524 I 3 Tth111 II 4 Bsp120 I 1 Fok I 22 NspB II 16 Xba I 2 Bsp1286 I 12 Fsp I 2 NspC I 3 Xca I 1 BspE I - Gdi II 4 Pac I - Xcm I 2 BspH I 5 Gsu I 6 PaeR7 I 1 Xho I 1 BspM I 2 Hae I 9 PflM I 3 Xma I 3 BspW I 31 Hae II 10 Ple I 6 Xmn I 2 Bsr I 22 Hae III 27
1 80 CTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGT GAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAA AAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAG AGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGGTACCCGGGTCGAGTA GGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGT TTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCCCGAATTCGAGCTCGGTACCCGGGGATCCTCTAG AGGATCCCCGGGATCGAAAGAGCCTGCTAAAGCAAAAAAGAAGTCACCATGTCGTTTACTTTGACCAACAAGAACGTGAT TTTCGTTGCCGGTCTGGGAGGCATTGGTCTGGACACCAGCAAGGAGCTGCTCAAGCGCGATCCCGTCGTTTTACAACGTC GTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCTTTATAGCGAA GAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGA AGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACG GTTACGATGCGCCCATCTACACCAACGTAACCTATTCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACG GGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAA CTTGGCGTTTCATCTGTGGTGCAACGTGCGCTGGGTCGGTTACGGCCAGGACAGTCGTTTGCCGTCTGAATTTGACCTGA GCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGTTGGAGTGACGGCAGTTATCTGGAAGATCAG GATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACTACACAAATCAGCGATTTCCATGT TGCCACTCGCTTTAATGATGATTTCAGCCGCGCTGAACTGGAGGCTGAAGTTCAGATGTGCGGCGAGTTGCGTGACTACC TACGGGTAACAGTTTCTTTATGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGAT GAGCGTGGTGGTTATGCCGATCGCGTCACACTACGTCTGAACGTCGAAAACCCGAAACTGTGGAGCGCCGAAATCCCGAA TCTCTATCGTGCGGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGATGTCGGTTTCCGCG AGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGAGGCGTTAACCGTCACGAGCATCAT CCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGT GCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCA ATATTGAAACCCACGGCATGGTGCCAATGAATCTGCTGACCGATGATCCGCGCTGGCTACCGGCGATGAGCGAACGCGTA ACGCGAATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAA TCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTGCAGTATGAAGGCGGCGGAGCCGACACCA CGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCTGTGCCGAAATGGTCCATC AAAAAATGGCTTTCGCTACCTGGAGAGACGCGCCCGCTGATCCTTTGCGAATACGCCCACGCGATGGGTAACAGTCTTGG CGGTTTCGCTAAATACTGGCAGGCGTTTCGTCAGTATCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGT CGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACCATCGCCAG TTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCAGCGCTGACGGAAGCAAAACACCAGCAGCAGTTTTT CCAGTTCCGTTTATCCGGGCAAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGCTCCTGCACT GGATGGTGGCGCTGGATGGTAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTCGCTCCACAAGGTAAACAGTTGATT GAACTGCCTGAACTACCGCAGCCGGAGAGCGCCGGGCAACTCTGGCTCACAGTACGCGTAGTGCAACCGAACGCGACCGG ATGGTCAGAAGCCGGGCACATCAGCGCCTGGCAGCAGTGGCGTCTGGCGGAAAACCTCAGTGTGACGCTCCCCGCCGCGT CCCACGCCATCCCGCATCTGACCACCAGCGAAATGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGC CAGTCAGGCTTTCTTTCACAGCTGTGGATTGGCGATAAAAAACAACTGCTGACGCCGCTGCGCGATCAGTTCACCCGTGC ACCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGG GCCATTACCAGGCCGAAGCAGCGTTGTTGCAGTGCACGGCAGATACACTTGCTGATGCGGTGCTGATTACGACCGCTCAC GCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGTAGTGGTCAAATGGCGATTAC CGTTGATGTTGAAGTGGCGAGCGATACACCGCATCCGGCGCGGATTGGCCTGAACTGCCAGCTGGCGCAGGTAGCAGAGC GGGTAAACTGGCTCGGATTAGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCCGCCTGTTTTGACCGCTGGGATCTG CCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGG CCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGTCAACAGCAACTGATGGAAACCAGCCATCGCC ATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGC CCGTCAGTATCGGCGGAATTACAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAATAATAACC GGCAGGCCATGTCTGAAAGTATTCGCGTAAGGAAATCCATTATGTACTATTTAAAAAACACAAACTTTTGGATGTTCGGT TTATTCTTTTTCTTTTACTTTTTTATCATGGGAGCCTACTTCCCGTTTTTCCCGATTTGGCTACATGACATCAACCATAT GAGCAAAAGTGATACGGGTATTATTTTTGCCGCTATTTCTCTGTTGTCGCTATTATTCCAACCGCTGTTGGTCTGCTTTC TGACAAACTCGGCCTCGACTCTAGACTGAGAACTTCAGGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTAT TGAAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATG GACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTTTCATTTTCT GTAACTTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTCGTTTATTTGTCAGATTGTAAGT ACTTTCTCTAATCACTTTTTTTTCAAGGCAATCAGGGTAATTATATTGTACTTCAGCACAGTTTTAGAGAACAATTGTTA TAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTAC ATCCTGGTAATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTC CAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTGTTGTGC TGTCTCATCATTTTGGCAAAGAATTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCC CTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGAC TTCTGGGTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTGGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATG GGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGA ACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGA CTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTA CTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAACTCGACTGCATTAATG AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGG TCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGA AAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCG CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCG GGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGT GCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACT TATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGG TGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGT TGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT TTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTAT ATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCAT CCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATA CCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCC TGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAA CGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAG TAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCT TTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCA ATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC AAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCA CCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATA CTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCA TGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCA