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Vector details:

0001-0592: (592) rDNA spacer targeting fragment 5339-5358: (20) T7 promoter
0593-3116: (2524) Vector backbone (pUC/pGEM) 5359-5418: (60) Tet operator fragment
3117-3412: (296) T. brucei actin mRNA processing 3' 5419-6231: (813) LacZ 'stuffer'
3413-4435: (1023) hygromycin B phosphotransferase ORF 6232-6318: (87) Tet operator fragment
4436-4570: (135) T. brucei actin mRNA processing 5' 6319-6338: (20) T7 promoter
4571-4865: (295) rDNA promoter 6339-6740: (402) T7 terminator x2 fragment
4866-5338: (473) T7 terminator x2 fragment  


p2T7TAblue is derived from p2T7TiB/GFP (LaCount et al., 2002). Digestion with Eam1105I generates 3´ overhanging T-ends which allow PCR products with 3´ A-ends to be cloned directly. Once inserted, RNAi targets are flanked by opposing T7 promoters with downstream operators. Recombinant vectors are linearised at the NotI site and targeted to rDNA loci in SMB (Single Marker Bloodstream-form) cells (Wirtz et al., 1999), that express T7 RNAP, Tet-repressor and the selectable marker, neomycin phosphotransferase (T7RNAP::TETR::NEO). Recombinants are selected with hygromycinB. Upon integration, the construct lies between rDNA spacer sequences. The operators bind Tet-repressor in the absence of tetracycline so dsRNA is only synthesised when tetracycline (1 μg/ml) is added to the growth medium. T7 terminators prevent transcription from extending into rDNA.
p2t7
for further information, or to obtain the plasmid, please contact David Horn david.horn@lshtm.ac.uk.
The following sequence can be downloaded here


GAATTCGATG CGAGGCGAAT CGCTCAGTAG TGTTAGTGCA GGCATTGTTT CTCTTTTCCA	60  
TTTCGGCTCG CTCTCTGTGC CAGAAAGGCT TTTGGAGTCT TGGTTACTCG TGGGACTAGG	120 
GCAAGCGTCG CTTCCCTAAT AATTGTTGCT AACGTTTCAC CGTGCGTGTG TGGATATCTT	180 
CCAAGTTGTC ATTTTCTCTA CCCCTCTCTT TTCCGGCGCG GTCTAACTTC CTCCAACTAG	240 
CGCATTTCCA TGATAACGGG GTCCACATAG TCCGTTGTAG TGAAATTGGC ATGCCATCAG	300 
GGCCAAGGCA TACATCGCGG CCGCGATAAA AATGATACGA TGATCTCCGT GTGGAGCAGA	360 
CCGTGGCGAC CAGTTTGTAC CATGGAAGTT CTTCGATAGA GTTATATCGA GTGCGGAATT	420 
GATGCTACGG TCACAGTGTC GTATCGTGTG GTCTCCCATG TGTTTTTGAC GGTGTACCAA	480 
TTATCTTTGC ACTAATGGGC TATGGAATCT TTCCTTTTAA TCAGGGGGAC ATGTTTATCC	540 
CACGCCAGTA GTCTGGAATT AGAATACATA CCAACTATAT GAGCTCGAAT TCTGCATTAA	600 
TGAATCGGCC AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG	660 
CTCACTGACT CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG	720 
GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA	780 
GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC	840 
CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA	900 
GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG	960 
ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT	1020
CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT	1080
GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG	1140
TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC	1200
AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC	1260
ACTAGAAGAA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA	1320
GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC	1380
AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG	1440
GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA	1500
AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT	1560
ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA	1620
GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTGGTGTA GATAACTACG	1680
ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA	1740
CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT	1800
CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT	1860
AGTTCGCCAG TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA	1920
CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA	1980
TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA	2040
AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT	2100
GTCATGCCAT CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA	2160
GAATAGTGTA TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG	2220
CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC	2280
TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA	2340
TCTTCAGCAT CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT	2400
GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT	2460
CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT	2520
ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC	2580
GTCTAAGAAA CCATTATTAT CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC	2640
TTTCGTCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA GCTCCCGGAG	2700
ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA	2760
GCGGGTGTTG GCGGGTGTCG GGGCTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG	2820
AGAGTGCACC ATTCGACGCT CTCCCTTATG CGACTCCTGC ATTAGGAAGC AGCCCAGTAG	2880
TAGGTTGAGG CCGTTGAGCA CCGCCGCCGC AAGGAATGGT GCAAGGAGAT GGCGCCCAAC	2940
AGTCCCCCGG CCACGGGGCC TGCCACCATA CCCACGCCGA AACAAGCGCT CATGAGCCCG	3000
AAGTGGCGAG CCCGATCTTC CCCATCGGTG ATGTCGGCGA TATAGGCGCC AGCAACCGCA	3060
CCTGTGGCGC CGGTGATGCC GGCCACGATG CGTCCGGCGT AGAGGATCTG GCTAGGAATA	3120
CTGCATAGAT AACAAACGCA TCAAACACAA CTGGTGGCAC TATTTCAATC ATGTCGACAC	3180
ACCAAGACGA GAAAATTTGA GTTAAACAAG ATGCCTACAA CCCTATATTT ATTATCTTCA	3240
AGTGATCTTC TCTTCTCTTG TGCGCTGTAC GTAAATGTGT TGCAAATAAG GCATAGGGCT	3300
GAGTACAGGC ACCACACGCG AGGAAAATCA TAAGCACTAG CCGTGTCAAC TCACAGCGAC	3360
TACAGAACAA TTTTGGCCAC ACAACCCGGT GTTAGGATCT CCGAGGCCTC TATTCCTTTG	3420
CCCTCGGACG AGTGCTGGGG CGTCGGTTTC CACTATCGGC GAGTACTTCT ACACAGCCAT	3480
CGGTCCAGAC GGCCGCGCTT CTGCGGGCGA TTTGTGTACG CCCGACAGTC CCGGCTCCGG	3540
ATCGGACGAT TGCGTCGCAT CGACCCTGCG CCCAAGCTGC ATCATCGAAA TTGCCGTCAA	3600
CCAAGCTCTG ATAGAGTTGG TCAAGACCAA TGCGGAGCAT ATACGCCCGG AGCCGCGGCG	3660
ATCCTGCAAG CTCCGGATGC CTCCGCTCGA AGTAGCGCGT CTGCTGCTCC ATACAAGCCA	3720
ACCACGGCCT CCAGAAGAAG ATGTTGGCGA CCTCGTATTG GGAATCCCCG AACATCGCCT	3780
CGCTCCAGTC AATGACCGCT GTTATGCGGC CATTGTCCGT CAGGACATTG TTGGAGCCGA	3840
AATCCGCGTG CACGAGGTGC CGGACTTCGG GGCAGTCCTC GGCCCAAAGC ATCAGCTCAT	3900
CGAGAGCCTG CGCGACGGAC GCACTGACGG TGTCGTCCAT CACAGTTTGC CAGTGATACA	3960
CATGGGGATC AGCAATCGCG CATATGAAAT CACGCCATGT AGTGTATTGA CCGATTCCTT	4020
GCGGTCCGAA TGGGCCGAAC CCGCTCGTCT GGCTAAGATC GGCCGCAGCG ATCGCATCCA	4080
TGGCCTCCGC GACCGGCTGC AGAACAGCGG GCAGTTCGGT TTCAGGCAGG TCTTGCAACG	4140
TGACACCCTG TGCACGGCGG GAGATGCAAT AGGTCAGGCT CTCGCTGAAT TCCCCAATGT	4200
CAAGCACTTC CGGAATCGGG AGCGCGGCCG ATGCAAAGTG CCGATAAACA TAACGATCTT	4260
TGTAGAAACC ATCGGCGCAG CTATTTACCC GCAGGACATA TCCACGCCCT CCTACATCGA	4320
AGCTGAAAGC ACGAGATTCT TCGCCCTCCG AGAGCTGCAT CAGGTCGGAG ACGCTGTCGA	4380
ACTTTTCGAT CAGAAACTTC TCGACAGACG TCGCGGTGAG TTCAGGCTTT TTCATCACTA	4440
GTTCTAGAGC TTATTTTATG GCAGCAACGA GACCTTACGT AAAGCAAAAC TAAATAAGAG	4500
CGGAGACTGC AATGCAGAGT AAAAAAAAAA AAAACATGAA TTTCAGAAGA CCTTGCTGTG	4560
CCCGGGCTCT GAGAGCGGTC AGTTGCTCTC CTGCTCCGTA CGCCGTAAGC GCTACTTTTA	4620
CTGCTTAATC TTTCCTATAT AAGATATATT TCTATTACTA TATATACTTT TTTAACTTTA	4680
TCATTATTAT CCTATTGTGT GTTGTATTGA CTTTACAATA AAACATATTA TATCTAACAT	4740
GTCAAAAGCA TCGGCTCTTC AATACAGTTA CCCGTCCCTT ATCAACACAG TACAATATAC	4800
TCTGTCATGA CAATAAGTAT ATATAAGAGC CAGAATGCAC CCGCGCTGGG TGGAAAGCTA	4860
GCTTGCATGC CTGCAGGGGC CCAATCCGGA TATAGTTCTC CTTTCAGCAA AAAACCCCTC	4920
AAGACCCGTT TAGAGGCCCC AAGGGGTTAT GCTAGTTATT GCTCAGCGGT GGCAGCAGCC	4980
AACTCAGCTT CCTTTCGGGC TTTGTTAGCA GCCCTATCCG GATATAGTTC TCCTTTCAGC	5040
AAAAAACCCC TCAAGACCCG TTTAGAGGCC CCAAGGGGTT ATGCTAGTTA TTGCTCAGCG	5100
GTGGCAGCAG CCAACTCAGC TTCCTTTCGG GCTTTGTTAG CAGCCTGCAG AATACTGCAT	5160
AGATAACAAA CGCATCAAAC ACAACTGGTG GCACTATTTC AATCATGTCG AGGAGGACCG	5220
AATACTAATA ACGACACTTG CGGTCAAAAA GTAGAAGAAC AAATGCTCAA CGATGAGTGA	5280
ATCAGGTTAG GGTAGTCGGA AAATTATACA GAATGTCTTT GGCAACACAC CGGCTGATTA	5340
ATACGACTCA CTATAGGGAG ATCTCCCTAT CAGTGATAGA GATCTCCCCG GGCCCCCCCT	5400
CGAGTTAACG ACCTTGCGTC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT	5460
CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT	5520
GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA	5580
CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC	5640
GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA	5700
GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG	5760
TCCATTCGCC ATTCAGGCTG CGCAACTGTT GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC	5820
TATTACGCCA GCTGGCGAAA GGGGGATGTG CTGCAAGGCG ATTAAGTTGG GTAACGCCAG	5880
GGTTTTCCCA GTCACGACGT TGTAAAACGA CGGCCAGTGA ATTGTAATAC GACTCACTAT	5940
AGGGCGAATT GGGCCCGACG TCGCATGCTC CCGGCCGCCA TGGCCGCGGG ATATCACTAG	6000
TGCGGCCGCC TGCAGGTCGA CCATATGGGA GAGCTCCCAA CGCGTTGGAT GCATAGCTTG	6060
AGTATTCTAT AGTGTCACCT AAATAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG	6120
TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA	6180
GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG CGTTGCGCGA CGCAAGGTCG	6240
TTAACGGATC CACTAGTTCT AGAGCGGCCG GCCGCCACCG CGGCTAGATC TCTATCACTG	6300
ATAGGGAGAT CTCCCTATAG TGAGTCGTAT TAATCAGCCG GTGTGTTGCC AAAGACATTC	6360
TGTATAATTT TCCAACTACC CTAACCTGAT TCACTCATCG TTGAGCATTT GTTCTTCTAC	6420
TTTTTGACCG CAAGTGTCGT TATTAGTATT CGGTCCTGGG CTGCAGGCTG CTAACAAAGC	6480
CCGAAAGGAA GCTGAGTTGG CTGCTGCCAC CGCTGAGCAA TAACTAGCAT AACCCCTTGG	6540
GGCCTCTAAA CGGGTCTTGA GGGGTTTTTT GCTGAAAGGA GAACTATATC CGGATAGGGC	6600
TGCTAACAAA GCCCGAAAGG AAGCTGAGTT GGCTGCTGCC ACCGCTGAGC AATAACTAGC	6660
ATAACCCCTT GGGGCCTCTA AACGGGTCTT GAGGGGTTTT TTGCTGAAAG GAGAACTATA	6720
TCCGGATTGG GCCCCTGCAG                                            	6741