Patent Application: US-3621793-A

Abstract:
what is described is a modified vector , such as a recombinant poxvirus , particularly recombinant vaccinia virus , having enhanced safety . the modified recombinant virus has nonessential virus - encoded genetic functions inactivated therein so that virus has attenuated virulence . in one embodiment , the genetic functions are inactivated by deleting an open reading frame encoding a virulence factor . in another embodiment , the genetic functions are inactivated by insertional inactivation of an open reading frame encoding a virulence factor . what is also described is a vaccine containing the modified recombinant virus having nonessential virus - encoded genetic functions inactivated therein so that the vaccine has an increased level of safety compared to known recombinant virus vaccines .

Description:
to develop the new vaccinia vaccine strain , nyvac ( vp866 ), the copenhagen vaccine strain of vaccinia virus was modified by the deletion of six nonessential regions of the genome encoding known or potential virulence factors . the sequential deletions are detailed below . all designations of vaccinia restriction fragments , open reading frames and nucleotide positions are based on the terminology reported in references 14 and 15 . the deletion loci were also engineered as recipient loci for the insertion of foreign genes . the regions sequentially deleted in nyvac are listed below . also listed are the abbreviations and open reading frame designations for the deleted regions ( 14 , 15 ) and the designation of the vaccinia recombinant ( vp ) containing all deletions through the deletion specified : ( 5 ) host range gene region ( c7l - k1l ) vp804 ; and dna cloning and synthesis . plasmids were constructed , screened and grown by standard procedures ( 33 , 46 , 47 ) . restriction endonucleases were obtained from bethesda research laboratories , gaithersburg , md ., new england biolabs , beverly , mass . ; and boehringer mannheim biochemicals , indianapolis , ind . klenow fragment of e . coli polymerase was obtained from boehringer mannheim biochemicals . bal - 31 exonuclease and phage t4 dna ligase were obtained from new england biolabs . the reagents were used as specified by the various suppliers . synthetic oligodeoxyribonucleotides were prepared on a biosearch 8750 or applied biosystems 380b dna synthesizer as previously described ( 44 ). dna sequencing was performed by the dideoxy - chain termination method ( 50 ) using sequenase ( 58 ) as previously described ( 17 ). dna amplification by polymerase chain reaction ( pcr ) for sequence verification ( 11 ) was performed using custom synthesized oligonucleotide primers and geneamp dna amplification reagent kit ( perkin elmer cetus , norwalk , conn .) in an automated perkin elmer cetus dna thermal cycler . excess dna sequences were deleted from plasmids by restriction endonuclease digestion followed by limited digestion by bal - 31 exonuclease and mutagenesis ( 32 ) using synthetic oligonucleotides . cells , virus , and transfection . the origins and conditions of cultivation of the copenhagen strain of vaccinia virus has been previously described ( 17 ). generation of recombinant virus by recombination , in situ hybridization of nitrocellulose filters and screening for b - galactosidase activity are as previously described ( 39 , 44 ). a better understanding of the present invention and of its many advantages will be had from the following examples , given by way of illustration . referring now to fig1 plasmid psd406 contains vaccinia hindiii j ( pos . 83359 - 88377 ) cloned into puc8 . psd406 was cut with hindiii and pvuii , and the 1 . 7 kb fragment from the left side of hindiii j cloned into puc8 cut with hindiii / smai , forming psd447 . psd447 contains the entire gene for j2r ( pos . 83855 - 84385 ). the initiation codon is contained within an nlaiii site and the termination codon is contained within an sspi site . direction of transcription is indicated by an arrow in fig1 . to obtain a left flanking arm , a 0 . 8 kb hindiii / ecori fragment was isolated from psd447 , then digested with nlaiii and a 0 . 5 kb hindiii / nlaiii fragment isolated . annealed synthetic oligonucleotides mpsyn43 / mpsyn44 ## str1 ## were ligated with the 0 . 5 kb hindiii / nlaiii fragment into puc18 vector plasmid cut with hindiii / ecori , generating plasmid psd449 . to obtain a restriction fragment containing a vaccinia right flanking arm and puc vector sequences , psd447 was cut with sspi ( partial ) within vaccinia sequences and hindiii at the puc / vaccinia junction , and a 2 . 9 kb vector fragment isolated . this vector fragment was ligated with annealed synthetic oligonucleotides mpsyn45 / mpsyn46 ## str2 ## to combine the left and right flanking arms into one plasmid , a 0 . 5 kb hindiii / smai fragment was isolated from psd449 and ligated with psd459 vector plasmid cut with hindiii / smai , generating plasmid psd460 . psd460 was used as donor plasmid for recombination with wildtype parental vaccinia virus copenhagen strain vc - 2 . 32 p labelled probe was synthesized by primer extension using mpsyn45 as template and the complementary 20mer oligonucleotide mpsyn47 ( 5 &# 39 ; ttagttaattaggcggccgc 3 &# 39 ;) as primer . recombinant virus vp410 was identified by plaque hybridization . referring now to fig2 plasmid psd419 contains vaccinia sali g ( pos . 160 , 744 - 173 , 351 ) cloned into puc8 . psd422 contains the contiguous vaccinia sali fragment to the right , sali j ( pos . 173 , 351 - 182 , 746 ) cloned into puc8 . to construct a plasmid deleted for the hemorrhagic region , u , b13r - b14r ( pos . 172 , 549 - 173 , 552 ), psd419 was used as the source for the left flanking arm and psd422 was used as the source of the right flanking arm . the direction of transcription for the u region is indicated by an arrow in fig2 . to remove unwanted sequences from psd419 , sequences to the left of the ncoi site ( pos . 172 , 253 ) were removed by digestion of psd419 with ncoi / smai followed by blunt ending with klenow fragment of e . coli polymerase and ligation generating plasmid psd476 . a vaccinia right flanking arm was obtained by digestion of psd422 with hpai at the termination codon of b14r and by digestion with nrui 0 . 3 kb to the right . this 0 . 3 kb fragment was isolated and ligated with a 3 . 4 kb hincii vector fragment isolated from psd476 , generating plasmid psd477 . the location of the partial deletion of the vaccinia u region in psd477 is indicated by a triangle . the remaining b13r coding sequences in psd477 were removed by digestion with clai / hpai , and the resulting vector fragment was ligated with annealed synthetic oligonucleotides sd22mer / sd20mer ## str3 ## generating psd479 . psd479 contains an initiation codon ( underlined ) followed by a bamhi site . to place e . coli betagalactosidase in the b13 - b14 ( u ) deletion locus under the control of the u promoter , a 3 . 2 kb bamhi fragment containing the betagalactosidase gene ( 52 ) was inserted into the bamhi site of psd479 , generating psd479bg . psd479bg was used as donor plasmid for recombination with vaccinia virus vp410 . recombinant vaccinia virus vp533 was isolated as a blue plaque in the presence of chromogenic substrate x - gal . in vp533 the b13r - b14r region is deleted and is replaced by beta - galactosidase . to remove beta - galactosidase sequences from vp533 , plasmid psd486 , a derivative of psd477 containing a polylinker region but no initiation codon at the u deletion junction , was utilized . first the clai / hpai vector fragment from psd477 referred to above was ligated with annealed synthetic oligonucleotides sd42mer / sd40mer ## str4 ## generating plasmid psd478 . next the ecori site at the puc / vaccinia junction was destroyed by digestion of psd478 with ecori followed by blunt ending with klenow fragment of e . coli polymerase and ligation , generating plasmid psd478e - . psd478e - was digested with bamhi and hpai and ligated with annealed synthetic oligonucleotides hem5 / hem6 ## str5 ## generating plasmid psd486 . psd486 was used as donor plasmid for recombination with recombinant vaccinia virus vp533 , generating vp553 , which was isolated as a clear plaque in the presence of x - gal . referring now to fig3 psd414 contains sali b cloned into puc8 . to remove unwanted dna sequences to the left of the a26l region , psd414 was cut with xbai within vaccinia sequences ( pos . 137 , 079 ) and with hindiii at the puc / vaccinia junction , then blunt ended with klenow fragment of e . coli polymerase and ligated , resulting in plasmid psd483 . to remove unwanted vaccinia dna sequences to the right of the a26l region , psd483 was cut with ecori ( pos . 140 , 665 and at the puc / vaccinia junction ) and ligated , forming plasmid psd484 . to remove the a26l coding region , psd484 was cut with ndei ( partial ) slightly upstream from the a26l orf ( pos . 139 , 004 ) and with hpai ( pos . 137 , 889 ) slightly downstream from the a26l orf . the 5 . 2 kb vector fragment was isolated and ligated with annealed synthetic oligonucleotides ati3 / ati4 ## str6 ## reconstructing the region upstream from a26l and replacing the a26l orf with a short polylinker region containing the restriction sites bglii , ecori and hpai , as indicated above . the resulting plasmid was designated psd485 . since the bglii and ecori sites in the polylinker region of psd485 are not unique , unwanted bglii and ecori sites were removed from plasmid psd483 ( described above ) by digestion with bglii ( pos . 140 , 136 ) and with ecori at the puc / vaccinia junction , followed by blunt ending with klenow fragment of e . coli polymerase and ligation . the resulting plasmid was designated psd489 . the 1 . 8 kb clai ( pos . 137 , 198 )/ ecorv ( pos . 139 , 048 ) fragment from psd489 containing the a26l orf was replaced with the corresponding 0 . 7 kb polylinker - containing clai / ecorv fragment from psd485 , generating psd492 . the bglii and ecori sites in the polylinker region of psd492 are unique . a 3 . 3 kb bglii cassette containing the e . coli betagalactosidase gene ( 52 ) under the control of the vaccinia 11 kda promoter ( 4 , 42 ) was inserted into the bglii site of psd492 , forming psd493kbg . plasmid psd493kbg was used in recombination with rescuing virus vp553 . recombinant vaccinia virus , vp581 , containing beta - galactosidase in the a26l deletion region , was isolated as a blue plaque in the presence of x - gal . to generate a plasmid for the removal of betagalactosidase sequences from vaccinia recombinant virus vp581 , the polylinker region of plasmid psd492 was deleted by mutagenesis ( 32 ) using synthetic oligonucleotide mpsyn177 ( 5 &# 39 ; aaaatgggcgtggattgttaactttatataacttattttttgaatatac 3 &# 39 ;). in the resulting plasmid , pmp494a , vaccinia dna encompassing positions [ 137 , 889 - 138 , 937 ], including the entire a26l orf is deleted . recombination between the pmp494δ and the beta - galactosidase containing vaccinia recombinant , vp581 , resulted in vaccinia deletion mutant vp618 , which was isolated as a clear plaque in the presence of x - gal . referring now to fig4 vaccinia sali g restriction fragment ( pos . 160 , 744 - 173 , 351 ) crosses the hindiii a / b junction ( pos . 162 , 539 ). psd419 contains vaccinia sali g cloned into puc8 . the direction of transcription for the hemagglutinin ( ha ) gene is indicated by an arrow in fig4 . vaccinia sequences derived from hindiii b were removed by digestion of psd419 with hindiii within vaccinia sequences and at the puc / vaccinia junction followed by ligation . the resulting plasmid , psd456 , contains the ha gens , a56r , flanked by 0 . 4 kb of vaccinia sequences to the left and 0 . 4 kb of vaccinia sequences to the right . a56r coding sequences were removed by cutting psd456 with rsai ( partial ; pos . 161 , 090 ) upstream from a56r coding sequences , and with eagi ( pos . 162 , 054 ) near the end of the gens . the 3 . 6 kb rsai / eagi vector fragment from psd456 was isolated and ligated with annealed synthetic oligonucleotides mpsyn59 - 62 ## str7 ## reconstructing the dna sequences upstream from the a56r orf and replacing the a56r orf with a polylinker region as indicated above . the resulting plasmid is psd466 . the vaccinia deletion in psd466 encompasses positions [ 161 , 185 - 162 , 053 ]. the site of the deletion in psd466 is indicated by a triangle in fig4 . a 3 . 2 kb bglii / bamhi ( partial ) cassette containing the e . coli beta - galactosidase gene ( 52 ) under the control of the vaccinia 11 kda promoter ( 4 , 17 ) was inserted into the bglii site of psd466 , forming psd466kbg . plasmid psd466kbg was used in recombination with rescuing virus vp618 . recombinant vaccinia virus , vp708 , containing beta - galactosidase in the a56r deletion , was isolated as a blue plaque in the presence of x - gal . beta - galactosidase sequences were deleted from vp708 using donor plasmid psd467 . psd467 is identical to psd466 , except that ecori , smai and bamhi sites were removed from the puc / vaccinia junction by digestion of psd466 with ecori / bamhi followed by blunt ending with klenow fragment of e . coli polymerase and ligation . recombination between vp708 and psd467 resulted in recombinant vaccinia deletion mutant , vp723 , which was isolated as a clear plaque in the presence of x - gal . referring now to fig5 the following vaccinia clones were utilized in the construction of pmpcskiδ . psd420 is sali h cloned into pucs . psd435 is kpni f cloned into puc18 . psd435 was cut with sphi and religated , forming psd451 . in psd451 , dna sequences to the left of the sphi site ( pos . 27 , 416 ) in hindiii m are removed ( 42 ). psd409 is hindiii m cloned into puc8 . to provide a substrate for the deletion of the [ c7lk1l ] gene cluster from vaccinia , e . coli beta - galactosidase was first inserted into the vaccinia m2l deletion locus ( 18 ) as follows . to eliminate the bglii site in psd409 , the plasmid was cut with bglii in vaccinia sequences ( pos . 28 , 212 ) and with bamhi at the puc / vaccinia junction , then ligated to form plasmid pmp409b . pmp409b was cut at the unique sphi site ( pos . 27 , 416 ). m2l coding sequences were removed by mutagenesis ( 18 , 32 ) using synthetic oligonucleotide ## str8 ## the resulting plasmid , pmp409d , contains a unique bglii site inserted into the m2l deletion locus as indicated above . a 3 . 2 kb bamhi ( partial )/ bglii cassette containing the e . coli betagalactosidase gene ( 52 ) under the control of the 11 kda promoter ( 4 ) was inserted into pmp409d cut with bglii . the resulting plasmid , pmp409dbg ( 18 ), was used as donor plasmid for recombination with rescuing vaccinia virus vp723 . recombinant vaccinia virus , vp784 , containing beta - galactosidase inserted into the m2l deletion locus , was isolated as a blue plaque in the presence of x - gal . a plasmid deleted for vaccinia genes [ c7l - k1l ] was assembled in puc8 cut with smai , hindiii and blunt ended with klenow fragment of e . coli polymerase . the left flanking arm consisting of vaccinia hindiii c sequences was obtained by digestion of psd420 with xbai ( pos . 18 , 628 ) followed by blunt ending with klenow fragment of e . coli polymerase and digestion with bglii ( pos . 19 , 706 ). the right flanking arm consisting of vaccinia hindiii k sequences was obtained by digestion of psd451 with bglii ( pos . 29 , 062 ) and ecorv ( pos . 29 , 778 ). the resulting plasmid , pmp581ck is deleted for vaccinia sequences between the bglii site ( pos . 19 , 706 ) in hindiii c and the bglii site ( pos . 29 , 062 ) in hindiii k . the site of the deletion of vaccinia sequences in plasmid pmp581ck is indicated by a triangle in fig5 . to remove excess dna at the vaccinia deletion junction , plasmid pmp581ck , was cut at the ncoi sites within vaccinia sequences ( pos . 18 , 811 ; 19 , 655 ), treated with bal - 31 exonuclease and subjected to mutagenesis ( 32 ) using synthetic oligonucleotide mpsyn233 5 &# 39 ; tgtcatttaacactatactcatattaataaaaataatatttatt 3 &# 39 ;. the resulting plasmid , pmpcsk1δ , is deleted for vaccinia sequences positions 18 , 805 - 29 , 108 , encompassing 12 vaccinia open reading frames [ c7l - k1l ]. recombination between pmpcsk1δ and the beta - galactosidase containing vaccinia recombinant , vp784 , resulted in vaccinia deletion mutant , vp804 , which was isolated as a clear plaque in the presence of x - gal . construction of plasmid psd548 for deletion of large subunit , ribonucleotide reductase ( i4l ) referring now to fig6 plasmid psd405 contains vaccinia hindiii i ( pos . 63 , 875 - 70 , 367 ) cloned in pucs . psd405 was digested with ecorv within vaccinia sequences ( pos . 67 , 933 ) and with smai at the puc / vaccinia junction , and ligated , forming plasmid psd518 . psd518 was used as the source of all the vaccinia restriction fragments used in the construction of psd548 . the vaccinia i4l gene extends from position 67 , 371 - 65 , 059 . direction of transcription for i4l is indicated by an arrow in fig6 . to obtain a vector plasmid fragment deleted for a portion of the i4l coding sequences , psd518 was digested with bamhi ( pos . 65 , 381 ) and hpai ( pos . 67 , 001 ) and blunt ended using klenow fragment of e . coli polymerase . this 4 . 8 kb vector fragment was ligated with a 3 . 2 kb smai cassette containing the e . coli beta - galactosidase gene ( 52 ) under the control of the vaccinia 11 kda promoter ( 4 , 42 ), resulting in plasmid psd524kbg . psd524kbg was used as donor plasmid for recombination with vaccinia virus vp804 . recombinant vaccinia virus , vp855 , containing beta - galactosidase in a partial deletion of the i4l gene , was isolated as a blue plaque in the presence of x - gal . to delete beta - galactosidase and the remainder of the i4l orf from vp855 , deletion plasmid psd548 was constructed . the left and right vaccinia flanking arms were assembled separately in puc8 as detailed below and presented schematically in fig6 . to construct a vector plasmid to accept the left vaccinia flanking arm , puc8 was cut with bamhi / ecori and ligated with annealed synthetic oligonucleotides 518a1 / 518a2 ## str9 ## forming plasmid psd531 . psd531 was cut with rsai ( partial ) and bamhi and a 2 . 7 kb vector fragment isolated . psd518 was cut with bglii ( pos . 64 , 459 )/ rsai ( pos . 64 , 994 ) and a 0 . 5 kb fragment isolated . the two fragments were ligated together , forming psd537 , which contains the complete vaccinia flanking arm left of the i4l coding sequences . to construct a vector plasmid to accept the right vaccinia flanking arm , puc8 was cut with bamhi / ecori and ligated with annealed synthetic oligonucleotides 518b1 / 518b2 ## str10 ## forming plasmid psd532 . psd532 was cut with rsi ( partial )/ ecori and a 2 . 7 kb vector fragment isolated . psd518 was cut with rsai within vaccinia sequences ( pos . 67 , 436 ) and ecori at the vaccinia / puc junction , and a 0 . 6 kb fragment isolated . the two fragments were ligated together , forming psd538 , which contains the complete vaccinia flanking arm to the right of i4l coding sequences . the right vaccinia flanking arm was isolated as a 0 . 6 kb ecori / bglii fragment from psd538 and ligated into psd537 vector plasmid cut with ecori / bglii . in the resulting plasmid psd539 , the i4l orf ( pos . 65 , 047 - 67 , 386 ) is replaced by a polylinker region , which is flanked by 0 . 6 kb vaccinia dna to the left and 0 . 6 kb vaccinia dna to the right , all in a puc background . the site of deletion within vaccinia sequences is indicated by a triangle in fig6 . to avoid possible recombination of beta - galactosidase sequences in the puc - derived portion of psd539 with beta - galactosidase sequences in recombinant vaccinia virus vp855 , the vaccinia i4l deletion cassette was moved from psd539 into prc11 , , a puc derivative from which all beta - galactosidase sequences have been removed and replaced with a polylinker region ( 9 ). psd539 was cut with ecori / psti and the 1 . 2 kb fragment isolated . this fragment was ligated into prc11 , cut with ecori / psti ( 2 . 35 kb ), forming psd548 . recombination between psd548 and the beta - galactosidase containing vaccinia recombinant , vp855 , resulted in vaccinia deletion mutant vp866 , which was isolated as a clear plaque in the presence of x - gal . dna from recombinant vaccinia virus vp866 was analyzed by restriction digests followed by electrophoresis on an agarose gel . the restriction patterns were as expected . polymerase chain reactions ( pcr ) ( 11 ) using vp866 as template and primers flanking the six deletion loci detailed above produced dna fragments of the expected sizes . sequence analysis of the pcr generated fragments around the areas of the deletion junctions confirmed that the junctions were as expected . recombinant vaccinia virus vp866 , containing the six engineered deletions as described above , was designated vaccinia vaccine strain &# 34 ; nyvac .&# 34 ; the gene encoding rabies glycoprotein g under the control of the vaccinia h6 promoter ( 61 , 62 ) was inserted into tk deletion plasmid psd513 . psd513 is identical to plasmid psd460 ( fig1 ) except for the presence of a polylinker region . referring now to fig7 the polylinker region was inserted by cutting psd460 with smai and ligating the plasmid vector with annealed synthetic oligonucleotides vq1a / vq1b ## str11 ## to form vector plasmid psd513 . psd513 was cut with smai and ligated with a smai ended 1 . 8 kb cassette containing the gene encoding the rabies glycoprotein g gene under the control of the vaccinia h6 promoter ( 61 , 62 ). the resulting plasmid was designated prw842 . prw842 was used as donor plasmid for recombination with nyvac rescuing virus ( vp866 ). recombinant vaccinia virus vp879 was identified by plaque hybridization using 32 p - 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:( i ) sequence characteristics :( a ) length : 28 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 2 : aattcccgggtagctagttaattacatg 28 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 73 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 3 : agcttcccgggtaagtaatacgtcaaggagaaaacgaaacgatc tgtagttagcggccgc60ctaattaactaat73 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 69 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 4 : attagttaattaggcggccgctaactacagatcgtttcgttttctccttgacgtattact60tacccggga69 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 5 : ttagttaattaggcggccgc20 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 22 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 6 : cgattactatgaaggatccgtt22 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 7 : aacggatccttcatagtaat20 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 41 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 8 : cgattactagatctgagctccccgggctcgagggatccgtt41 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 39 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 9 : aacggatccctcgagcccggggagctcagatctagtaat39 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 16 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 10 : gatccgaattctagct16 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 12 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 11 : ag ctagaattcg12 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 75 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 12 : tatgagtaacttaa ctcttttgttaattaaaagtatattcaaaaaataagttatataaat60agatctgaattcgtt75 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 73 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 13 : aacgaattcagatctatttatataacttattttttgaatatacttttaattaacaaaaga60gttaagttactca73 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 49 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 14 : aaaatgggcgtggattgttaactttatataacttattttttgaatatac49 ( 2 ) information for seq id no : 15 : ( i ) sequence characteristics :( a ) length : 67 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 15 : acacgaatgattttctaaagtatttggaaagttttataggtagttgatagaacaaaatac60ataattt 67 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 51 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 16 : tctatcaactacctataaaactttccaaatactttagaa aatcattcgtgt51 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 46 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 17 : tgtaaaaataaatcactttttatactaagatctcccgggctgcagc 46 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 66 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 18 : ggccgctgcagcccgggagatcttagtataaaaagtgatttatttttacaaaattatgta 60ttttgt66 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 50 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 19 : tttctg tatatttgcaccaatttagatcttactcaaaatatgtaacaata50 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 44 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 20 : tgtcatttaacactatac tcatattaataaaaataatatttatt44 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 21 : gatcctgagtactttgtaatataatgatat atattttcactttatctcatttgagaataa60aaagatcttagg72 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 22 : aattcctaagatctttttattctcaaatgagataaagtgaaaatatatatcattatatta60caaagtactcag72 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 23 : gatccagatctcccgggaaaaaaattatttaacttttcattaatagggatttgacgtatg60tagcgtactagg 72 ( 2 ) information for seq id no : 24 :( i ) sequence characteristics :( a ) length : 72 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 24 : aattcctagtacgcatcatacgtcaaatccctattaatgaaa agttaaataatttttttc60ccgggagatctg72 ( 2 ) information for seq id no : 25 :( i ) sequence characteristics :( a ) length : 40 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 25 : gggagatctctcgagctgcagggcgccggatcctttttct40 ( 2 ) information for seq id no : 26 :( i ) sequence characteristics :( a ) length : 40 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 26 : agaaaaaggatccggcgccctgcagctcgagagatctccc40