7/7/2000
pXGLeishmania expression vector with NEO marker
Strain B1288
First pXG-series vector with Tn5Kanmarker (= NEO to eukaryote world). Sometimes to distinguish from later vectors we refer to it as pXGNEO or pXG(NEO).
Leishmania intergenic regions, providing signals for trans-splicing and polyadenylation, flank the unique polylinker SmaI or BamHI sites. Insertion of ORF into these sites gives high level of expression, fairly constant through parasite life cycle (log, stationary phase promastigotes; lesion amastigotes).
Any site in the vector (compatible with marker function) can be used for shuttle vector purposes.
The reference for pXG(NEO) is:
Ha, D.S., Schwarz, J.K., S.J. Turco, and S.M. Beverley (1996), "Use of the green fluorescent protein as a marker in transfected Leishmania ", Mol. Biochem. Parasitol. 77: 57-64.
This reference shows data for pXG-mediated β-galactosidase expression throughout the life cycle.
Chakkalath, H.R., A.A. Siddiqui, A.H. Shankar, D.E. Dobson, S.M. Beverley, and R.G. Titus (2000), Priming of a -galactosidase (GAL)-specific type 1 response in BALB/c mice infected with GAL-transfected Leishmania major", Infection & Immunity 68: 809-814.
The NEO gene is originally from pR-NEO (Kapler, G.M., C.M. Coburn, and S.M. Beverley (1990), "Stable Transfection of the human parasite Leishmania delineates a 30 kb region sufficient for extra-chromosomal replication and expression", Mol. Cell. Biol.10: 1084-1094.)
Notice that the marker is actually a mutant KAN gene (Yenofsky, R. L. Fine, M., Pellow, J. W.; 1990; “A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure”, PNAS 87: 3435-9). Our experience is that it works well and perhaps even better than wild-type in Leishmania. Perhaps the effect is species-specific (due to protein stability?).
Revisions:
1/27/01 – sequence around ‘missing’ BglII site determined.
Sequence (assembled from previous files and not always experimental).
LOCUS pXG 6820 bp DNA CIRCULAR SYN 27-Jan-2001
DEFINITION constitutive pX series vector
REFERENCE 1 (bases 1 to 6820)
AUTHORS Stephen M. Beverley
FEATURES Location/Qualifiers
CDS 1288..1363
/gene="5'DST"
/product="5'DST"
CDS 1414..1530
/gene="6.2-l"
/product="6.2-l"
CDS 2000..2152
/gene="1.7-l"
/product="1.7-l"
misc_feature complement (2182)
/gene="T3"
/product="T3"
CDS complement (3182..3942)
/gene="amp"
/product="amp"
misc_feature 4906
/gene="SP6"
/product="SP6"
CDS 4970..5169
/gene="3'13K"
/product="3'13K"
CDS 5664..879
/gene="NEO/DHFR"
/product="NEO/DHFR-TS"
BASE COUNT 1448 a 2010 c 1826 g 1536 t
ORIGIN
1 ACTAGTGATC CCGCCCGCAC CCGCGCGCGC TCAGGCCGCG TGTCGTCGTG CTCTTTCCAT
61 TTTTTTTTTG TGTGTGGTGG TGATTTGAGC TGCTCTCCGT TGTGTGCTGG GGACCCTCCT
121 TCCCTCGATC TCCTCGTGCG GGGTCTCCGA CGCGCAGACG CGGTGCGTGC GAGCGTGCAC
181 GTGCTGTCGC CGTGGCTGTA GTTGCGAGCG GAGAGAGAGA GAGAGGGAGG GAGGGAGGGC
241 AGAGGGCAGA GGACATGCGG GTGGGAACGT GCACCGGCCT CGTCTCACGC AGCTGGAGCC
301 CACGAATGCC ACCACCACAC CCTTCTTCCC CCCCCCTCCC TCTTCTTCCC ACGGCGGCGA
361 CGACGACGGG GGCTCAGCTC ACGCTTCTGT AGGGTATTAT ATTAAAGCAC ATGTGCGTGC
421 TGTGCTTCGG CTTCTTGTTG GTGGTCGTTC GCTTTCAAGT CCACGACTCC TGCCGTTGCT
481 CACCGCCGGG CTGTCTTCTT TCGCCCCTCC CTCGGTGCCT CTTCCGCCGC CGCACGCGTG
541 CGCTGATCAC GCGTCTTGCG TGATGTGCCT GTGTGTATAC ACACCGTGCA CGGAGAGAGC
601 GAGCGAGCGA GAGAGAGAGA GGGCGAGAGG AAGAATGGCC GGTGCCTCGC GTGGGCAAGC
661 GTGCGCGAGT TTGTCGTGTC GCTGCGCCGC GTGGATGTCG CGCAGCGCAA GACCCCGTGC
721 GCCTGAAACG TGAAGGGAGG TCGAGAAGCG TGCCGCATGA GGCCTTAAGG CAGGAAAGTG
781 AAAAGAGCTC GACGCGAGTG CAGCACGCGC GACATCGAAC AGGACGGAAT AGACACTCAG
841 CCCCTTCCCC TTTCAAAAAC TGAATGGACG GACATCGTAA CGCGCTTCTC TCCCCTCCAC
901 TCCCCCTGTC CCTGTCTCCT TGACGTGTGC TGCTCTCGTG AGCGACAGAT GAAGCGAGGC
961 GTAGAAGGAA AGGGGGGGGA GCGAGGCCTG CACACACACA CACACACACA CACACACACA
1021 CACACACACA CACACACACA CACACACGAA AAGCAAGTTT GAGGCAGACG TGGTTTCACA
1081 CGATTCCTGT GCAAGAAGCG TGCCTGCGTG ACGGTGTGGC TGTGTATGCA TGCGTGAAGG
1141 GTTACCGCAA CGATGCGCAA TGGACTCCCC CGCTTTCCAT TTCGTCACCT TCCGCCTCTC
1201 TCTCTCTCTC TCTCACCATC TACGCGTGCA CATCATCAAC TGTCTCTTGT CGGTGCTCAC
1261 CACCCTCAAC CACCCCTCAC TTTCAAGGCT TCCCGAACGC ACACAAAAGG CGTGAAAACC
1321 GCTCGCGTGT GTTGAGCCGT CCACCGTAGC CCTCCCCCTG TCCCCGGGGG ATCCACTAGT
1381 TCTAGTTCTA GGGGGCGAGC TCGAATTCAG ATCCTCGTGT GAGCGTTCGC GGAATCGGTC
1441 GCTCGTGTTT ATGCCCGTCT TGGTGTTGTG CTCGCAAGGC GGTGCAGCAG GATACCGTCG
1501 CCCTCCTCTC TCCTTGCTTC TCTGTTCTTC AATTCGCGAT CTCACAGAGG CCGGCTGTGC
1561 ACGCCCTTCC TCACCCTCCT TTTCCCACCT CTCGGCCACC GGTCGGCTCC GTTCCGTCTG
1621 CCGTCGAGAA GGGACGGGCA TGTGCAGCTC CTCCCTTTCT CTCGCGCGCG CATCTTCTCT
1681 TGCTTGTGGC ACTCACGCTC ATGCGTCAAG GCGGCCCCAC GCGAGCCCCT GCGCTCCCTT
1741 CCCTCTTGCG CATCCGTAGC CGGACTGGTC GATGCGCAAG GCCGGCATGA AGGAGCGCGT
1801 GCCCTCAAGA GCGGACTATC ATGCCCTACG TGGGCCACGC AGCGATGAGG CCGGCTTCGC
1861 GGAGATGCGT CACGCACGTG CCAGATGATG CCGTACGCCT TCCTTGACTT GCGCCCCCCT
1921 CTCTTCCTCC GTCTCTCACT CTCTCTCTCT CACACACACA CACACACACA CACACACACA
1981 AAGCTTCCTT GCACACAACG TCCCCCTCGT TGTGCGGGCT GACGCCTCCC GTTGACAACT
2041 TGCCTTACGC ACGCAGCACT CGGCCAGCCA ACGAGGCCGC AGCGGCATCA CGTGTATCTT
2101 TCCACAGAAA CCGCGTGCTT TCGGATACTC TAGTATTTTT GTTTGTGGTC GAATCTCCCT
2161 TTAGTGAGGG TTAATTAGTC CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT
2221 TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC
2281 TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG
2341 ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG
2401 CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC
2461 GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG
2521 GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT
2581 TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG CTGTAGGTAT CTCAGTTCGG
2641 TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT
2701 GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC
2761 TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT
2821 TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT ATCTGCGCTC
2881 TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA
2941 CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT
3001 CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC
3061 GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT
3121 AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC
3181 AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG
3241 CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG
3301 CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC
3361 CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA
3421 TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG
3481 TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT
3541 CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA
3601 GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG
3661 TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA
3721 CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT
3781 GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA
3841 TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT
3901 CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT
3961 CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA
4021 AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT
4081 GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC
4141 GCACATTTCC CCGAAAAGTG CCACCTGACG TCTAAGAAAC CATTATTATC ATGACATTAA
4201 CCTATAAAAA TAGGCGTATC ACGAGGCCCT TTCGTCTCGC GCGTTTCGGT GATGACGGTG
4261 AAAACCTCTG ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG
4321 GGAGCAGACA AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCTGGCTTA
4381 ACTATGCGGC ATCAGAGCAG ATTGTACTGA GAGTGCACCA TATCGACGCT CTCCCTTATG
4441 CGACTCCTGC ATTAGGAAGC AGCCCAGTAG TAGGTTGAGG CCGTTGAGCA CCGCCGCCGC
4501 AAGGAATGGT GCATGCAAGG AGATGGCGCC CAACAGTCCC CCGGCCACGG GGCCTGCCAC
4561 CATACCCACG CCGAAACAAG CGCTCATGAG CCCGAAGTGG CGAGCCCGAT CTTCCCCATC
4621 GGTGATGTCG GCGATATAGG CGCCAGCAAC CGCACCTGTG GCGCCGGTGA TGCCGGCCAC
4681 GATGCGTCCG GCGTAGAGGA TCTGGCTAGC GATGACCCTG CTGATTGGTT CGCTGACCAT
4741 TTCCGGGGTG CGGAACGGCG TTACCAGAAA CTCAGAAGGT TCGTCCAACC AAACCGACTC
4801 TGACGGCAGT TTACGAGAGA GATGATAGGG TCTGCTTCAG TAAGCCAGAT GCTACACAAT
4861 TAGGCTTGTA CATATTGTCG TTAGAACGCG GCTACAATTA ATACATAACC TTATGTATCA
4921 TACACATACG ATTTAGGTGA CACTATAGAA TACAAGCTTG GGCTGCAGGT CGACGGGGTG
4981 ATGGAGAGAA TGGTTGACCA TGAGGACGCC GATGGGCGAT GCGCCAGCGC GTGCTTGCGT
5041 ACTTCAATCG TTTTTCATCT TGATTCAGAG CTAAGTAGTA GCACACCACA GCAAGCGTAA
5101 TCATGTAGCA TGTATGCAGG TGCACGAGTA GGCACTCTCT GTCTCTGTCC GGCATCTATG
5161 ATGCTCTCCG GCCGCGTGCG CGCGACGCCT CTGTGTGAGT GCTCGTGTGA GTGTGTGAGA
5221 GCTGTCCAGG CCTTTCCTCT GCCTACTCGT CCCACGCTCA GGCAGGCACA GAGGAATGAA
5281 AAGGAAGACG AGGACGCAAC GCCAGGCGCA TCGACATCGC GCACGTGTTT GCGGTGTACT
5341 GTGCGTCGAT GATCGAGCGG CCAAGCGTCC AGCTTCACGC GAGGAGGACT GCATCGCTGC
5401 TCCTGTGCGC GTGCGTGCAC GCGTCAAGGC ACGCTCGATT CGTTCCCAGG GTGGATACCC
5461 TCCCCCCTCC CCCAGCTGCA GACAACCCCC ACCCTCTCGA GGCTTGCACC CAGGCTCGTC
5521 AGTCTCTCTA CCACTTTCTG CCTTCTGTAG TGCACCCCAC CCCTGCATTC TCCTCTCTCT
5581 CACACACACA CACACACACA CACACACACA CACACACACA CGTACACACG TACACACACA
5641 CGTGCGCATG CTTGACGCAT ACGCGACGAA TTCGAAAGCT CACCTCATTC CTCCCTCCTC
5701 ACACCATCAT CGGCATCCAT AGAGACACGT GCGCGTAGAA CGATACAGCT CACGCGCACA
5761 GAGAGGCGTG TTCTGGTCGT GCGCATCATC GGCACAGCAC TGGCGGACGA AACTCGCACA
5821 CAGGCACGCC GCCTCCTTTC ACCCGTCATA GATAGTTGAA TTAGACGCCC TCCTCCTCCC
5881 TCATCATCGC CGTCGTCATC CGGGTCCGAG CACTAGTCGC GTGTGGCCTC GAACACCGAG
5941 CGACCCTGCA GCCAATATGG GATCGGCCAT TGAACAAGAT GGATTGCACG CAGGTTCTCC
6001 GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTC
6061 TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGA
6121 CCTGTCCGGT GCCCTGAATG AACTGCAGGA CGAGGCAGCG CGGCTATCGT GGCTGGCCAC
6181 GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT
6241 GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA
6301 AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCC
6361 ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG AAGCCGGTCT
6421 TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGC
6481 CAGGCTCAAG GCGCGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTG
6541 CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT
6601 GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG CTGAAGAGCT
6661 TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCA
6721 GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GGGGATCGGC AATAAAAAGA
6781 CAGAATAAAA CGCACGGGTG TTGGGTCGTT TGTTCGGATC
//
B1288 pXG