21C4
Site of transposon insertion cloned as PstI fragment into pSP72. Sequenced from the outside using SP6 and T7, and then with specific oligos to give an assembled sequence: TTTTCGNCCC TGAAGCTTGC ATGCCTGCAG ATCGTGGTGA TCCCGATCGG CCTGGGCCTG ATCGTCCACC ATACCCTGAC CAAAACGGTG AAGCGCATCG AGCCGCTGCT GCCGGCGCTG TCGATGGTGT GCATTCTGGC CATCATCAGC GCGGTGGTGG CGGGCAGCCA AAGCCATATC GCCTCGGTCG GTCTGGTGGT GATCGTCGCG GTGATCCTGC ACAACGGCAT CGGCCTGCTG AGCGGCTACT GGGGCGGCAA ACTGTTCGGC TTCGACGAGT CCACCTGCCG CACGCTGGCG ATCGAAGTCG GGATGCAGAA CTCCGGGCTG GCAGCCACGT TGGGCAAAAT CTATTTCTCG CCGCTGGCCG CGCTGCGGGC GCGCTGTTCT CGGTCTGGCA CAACCTCTNC GGTCGCTGCT GCAGGGGATT TGGGTCCNGC CGCCCTATCA AGAAAAATAA ACCGCCTGTC GGGACGCAAA CAATTGCGTC CCGTTTCTTC CCTGCGAAAT TTCGTATTTC CCACTGGTCG GATTGTCATA ATTTCCCTCA TCCCCTATCA TCCGTGAAAA ATAAAAATTC AGATTCTTCC TATCCATGCC GATAATGTTG GCAAGAGGAA GAGACGTTCT ACTTTTTTTA ATTAATTAAG TCACCAGGGT TGCCCGCCAT AAAAGAAGCA GCCTAATTAT TATTTAGATA TATACCCACC GTCTTATTTG CTGCGGCAAA TCAAGATACC TCGCAATGTG CATGTCGCTC ATTATTTCGG GCCGGCCCTC GGCCTGACTC TTATACACAA GTGCGGCCGC GGCCTAGGCG GCCGAAGCTT GTCGACGAAT TCCCGGGGAT CCGGTGATTG ATTGAGCAAG CTTTATGCTT GTAAACCGTT TTGTGAAAAA ATTTTTAAAA TAAAAAAGGG GACCTCTAGG GTCCCCAATT AATTAGTAAT ATAATCTATT AAAGGTCATT CAAAAGGTCA TCCACCGGAT CAATTCCCCT GCTCGCGCAG GCTGGGTGCC AAGCTCTCGG GTAACATCAA GGCCCGATCC TTGGAGCCCT TGCCCTCCCG CACGATGATC GTGCCGTGAT CGAAATCCAG ATCCTTGACC CGCAGTTGCA AACCCTCACT GATCCGCACA TCGGGATCAC TTGTTCGGTG AGGTAGCAGT CGAAGTCGCA TTGCAGCGCA CGCTTGAGGA ACGGGGTGAG GCTGGTGCCT TCGGTGGAGC GCCACCGCGC CATGACCTGG ATGCAACGCC TCAAGCGAGT CTTCAATATT GATATTGAAG TCTGCGAACA CTGCGGCGGT CACGTCAAAG TGN
BLASTN with this sequence (full results here) gives:
>gi|340726|gb|M22618.1|SMASHLAB Serratia marcescens (clone pES2) hemolysin (hlsB), and hemolysin
determinant (hlsB) genes, complete cds
Length = 7253
Score = 777 bits (392), Expect = 0.0
Identities = 533/575 (92%), Gaps = 6/575 (1%)
Strand = Plus / Plus
Query: 213 gatcctgcacaacggcatcggcctgctgagcggctactggggcggcaaactgttcggctt 272
||||||||||||||||||||| |||||||||||||| ||||||||||| ||||||||||
Sbjct: 2 gatcctgcacaacggcatcggtctgctgagcggctattggggcggcaagttgttcggctt 61
Query: 273 cgacgagtccacctgccgcacgctggcgatcgaagtcgggatgcagaactccgggctggc 332
||||||||||||||||||||||||||||||||||||||| ||||||||||||||| ||||
Sbjct: 62 cgacgagtccacctgccgcacgctggcgatcgaagtcggcatgcagaactccgggttggc 121
Query: 333 agccacgttgggcaaaatctatttctcgccgctggccgcgctg-cgggcgcgctgttctc 391
|||||| |||| |||||||| ||||||||||||||||||||| ||||||||||||||||
Sbjct: 122 ggccacgctgggtaaaatctacttctcgccgctggccgcgctgccgggcgcgctgttctc 181
Query: 392 ggtctggcacaacctctncgg-tcgctgctgcaggggatttgggtccngccgccctatca 450
||||| ||||||||||| ||| ||||||||| ||| | | ||||| ||||||||||||
Sbjct: 182 ggtct-gcacaacctctccggttcgctgctg-gcgggttattggtccggccgccctatca 239
Query: 451 ag-aaaaataaaccgcctgtcgggacgcaaacaattgcgtcccgtttcttccctgcgaaa 509
|| |||||||||||||||| |||||| ||||||||||||||| ||||||| ||||||||
Sbjct: 240 agaaaaaataaaccgcctgcagggacg-aaacaattgcgtcccttttcttctctgcgaaa 298
Query: 510 tttcgtatttcccactggtcggattgtcataatttccctcatcccctatcatccgtgaaa 569
|||||||||||||||||||||||||||||||||||||| ||| |||||||||||||||||
Sbjct: 299 tttcgtatttcccactggtcggattgtcataatttcccccataccctatcatccgtgaaa 358
Query: 570 aataaaaattcagattcttcctatccatgccgataatgttggcaagaggaagagacgttc 629
|||||||| |||||||||||||||||||||||||||||||||||||||||||||||| ||
Sbjct: 359 aataaaaactcagattcttcctatccatgccgataatgttggcaagaggaagagacgctc 418
Query: 630 tacnnnnnnnaattaattaagtcaccagggttgcccgccataaaagaagcagcctaatta 689
|| ||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 419 cactttttttaattaattaagtcaccagggttgcccgccataaaagaagcagcctaatta 478
Query: 690 ttatttagatatatacccaccgtcttatttgctgcggcaaatcaagatacctcgcaatgt 749
|||||||||||||||||||||||||||||||||| |||||||||||||| ||||||||||
Sbjct: 479 ttatttagatatatacccaccgtcttatttgctgtggcaaatcaagatatctcgcaatgt 538
Query: 750 gcatgtcgctcattatttcgggccggccctcggcc 784
|||||||||||||||||||||||||||| ||||||
Sbjct: 539 gcatgtcgctcattatttcgggccggccgtcggcc 573
As well as
>gi|1163175|gb|U32991.1|ECU32991 Escherichia coli mini-Tn5 kanamycin transposon
Length = 2356
Score = 363 bits (183), Expect = 3e-98
Identities = 212/219 (96%), Gaps = 2/219 (0%)
Strand = Plus / Minus
Query: 918 ggggacctctagggtccccaattaattagtaatataatctattaaaggtcattcaaaagg 977
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 2222 ggggacctctagggtccccaattaattagtaatataatctattaaaggtcattcaaaagg 2163
Query: 978 tcatccaccggatcaattcccctgctcgcgcaggctgggtgccaagctctcgggtaacat 1037
||||||||||||||||||||||||||| | ||| |||||| |||||||||| ||||||||
Sbjct: 2162 tcatccaccggatcaattcccctgctcacacagactgggtaccaagctctc-ggtaacat 2104
Query: 1038 caaggcccgatccttggagcccttgccctcccgcacgatgatcgtgccgtgatcgaaatc 1097
||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||
Sbjct: 2103 caaggcccgatccttggagcccttgccctcccg-acgatgatcgtgccgtgatcgaaatc 2045
Query: 1098 cagatccttgacccgcagttgcaaaccctcactgatccg 1136
|||||||||||||| ||||||||||||||||||||||||
Sbjct: 2044 cagatccttgacccacagttgcaaaccctcactgatccg 2006
Score = 198 bits (100), Expect = 1e-48
Identities = 109/112 (97%)
Strand = Plus / Minus
Query: 784 ctgactcttatacacaagtgcggccgcggcctaggcggccgaagcttgtcgacgaattcc 843
||||||||||||||||||| ||||| ||||||||||||||| ||||||||||||||||||
Sbjct: 2356 ctgactcttatacacaagttcggccacggcctaggcggccgcagcttgtcgacgaattcc 2297
Query: 844 cggggatccggtgattgattgagcaagctttatgcttgtaaaccgttttgtg 895
||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 2296 cggggatccggtgattgattgagcaagctttatgcttgtaaaccgttttgtg 2245
Score = 149 bits (75), Expect = 1e-33
Identities = 75/75 (100%)
Strand = Plus / Plus
Query: 918 ggggacctctagggtccccaattaattagtaatataatctattaaaggtcattcaaaagg 977
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 114 ggggacctctagggtccccaattaattagtaatataatctattaaaggtcattcaaaagg 173
Query: 978 tcatccaccggatca 992
|||||||||||||||
Sbjct: 174 tcatccaccggatca 188
Score = 119 bits (60), Expect = 9e-25
Identities = 60/60 (100%)
Strand = Plus / Plus
Query: 836 cgaattcccggggatccggtgattgattgagcaagctttatgcttgtaaaccgttttgtg 895
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 32 cgaattcccggggatccggtgattgattgagcaagctttatgcttgtaaaccgttttgtg 91
Thus the transposon is inserted in a region of the Serrtaia marcescens genome that has previously been cloned: the shlBA operon. Relative to the pES2 clone, the transposon is inserted at nucleotide position 574. In pES2, the coding sequence of shlB, which encodes a hemolysin transporter, is at a position 635. In other words, the insertion is 61 bp upstream of the start of shlB. The downstream gene, shlA encodes a hemolysin.
The shlBA operon is one of the best characterised loci in Serratia marcescens and is known to be important for virulence. It has been extensively studied by the group of V. Braun; see, for example, this web page and the abstracts by Poole et al., Schonherr et al., Hertle et al..