IRESite_Id:132 characteristics of BCL2 IRES studied in pRL-BCL2-FL plasmid

IRESite: The database of experimentally verified IRES structures

The nucleic acid data:

IRESite Id: `132`	Version: `7`
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš	Last change: `2007-05-15 00:00:00`

IRESite record type:

plasmid_with_promoter_and_putative_IRES_translationally_characterized

The shape of the nucleic acid molecule translated:
linear The quality of the mRNA/+RNA sequence:
our_best_guess

The mRNA/+RNA description:

Bicistronic mRNA containing renilla and firefly luciferases with BCL2 IRES segment in the intercistronic
region. The mRNA is spliced and does not contain the chimeric intron. The sequence ends at its 3'-end right
after the poly(A) signal from bovine growth hormone (BGH) mRNA and thus the 3'-UTR might be slightly wrong (it
is unknown what region of the BGH signal was transcribed and where the poly(A) tail was added).

The mRNA/+RNA sequence represented in the +DNA notation:

TCAGATCACT AGAAGCTTTA TTGCGGTAGT TTATCACAGT TAAATTGCTA ACGCAGTCAG TGCTTCTGAC ACAACAGTCT CGAACTTAAG CTGCAGAAGT 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

TGGTCGTGAG GCACTGGGCA GGTGTCCACT CCCAGTTCAA TTACAGCTCT TAAGGCTAGA GTACTTAATA CGACTCACTA TAGGCTAGCC ACCATGACTT 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

CGAAAGTTTA TGATCCAGAA CAAAGGAAAC GGATGATAAC TGGTCCGCAG TGGTGGGCCA GATGTAAACA AATGAATGTT CTTGATTCAT TTATTAATTA 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

TTATGATTCA GAAAAACATG CAGAAAATGC TGTTATTTTT TTACATGGTA ACGCGGCCTC TTCTTATTTA TGGCGACATG TTGTGCCACA TATTGAGCCA 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

GTAGCGCGGT GTATTATACC AGACCTTATT GGTATGGGCA AATCAGGCAA ATCTGGTAAT GGTTCTTATA GGTTACTTGA TCATTACAAA TATCTTACTG 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

CATGGTTTGA ACTTCTTAAT TTACCAAAGA AGATCATTTT TGTCGGCCAT GATTGGGGTG CTTGTTTGGC ATTTCATTAT AGCTATGAGC ATCAAGATAA 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

GATCAAAGCA ATAGTTCACG CTGAAAGTGT AGTAGATGTG ATTGAATCAT GGGATGAATG GCCTGATATT GAAGAAGATA TTGCGTTGAT CAAATCTGAA 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

GAAGGAGAAA AAATGGTTTT GGAGAATAAC TTCTTCGTGG AAACCATGTT GCCATCAAAA ATCATGAGAA AGTTAGAACC AGAAGAATTT GCAGCATATC 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

TTGAACCATT CAAAGAGAAA GGTGAAGTTC GTCGTCCAAC ATTATCATGG CCTCGTGAAA TCCCGTTAGT AAAAGGTGGT AAACCTGACG TTGTACAAAT 
^801       ^811       ^821       ^831       ^841       ^851       ^861       ^871       ^881       ^891       

TGTTAGGAAT TATAATGCTT ATCTACGTGC AAGTGATGAT TTACCAAAAA TGTTTATTGA ATCGGACCCA GGATTCTTTT CCAATGCTAT TGTTGAAGGT 
^901       ^911       ^921       ^931       ^941       ^951       ^961       ^971       ^981       ^991       

GCCAAGAAGT TTCCTAATAC TGAATTTGTC AAAGTAAAAG GTCTTCATTT TTCGCAAGAA GATGCACCTG ATGAAATGGG AAAATATATC AAATCGTTCG 
^1001      ^1011      ^1021      ^1031      ^1041      ^1051      ^1061      ^1071      ^1081      ^1091      

TTGAGCGAGT TCTCAAAAAT GAACAATAAT TCTAGAGCGG CCGCAAAAAA TAAAACCCTC CCCCACCACC TCCTTCTCCC CACCCCTCGC CGCACCACAC 
^1101      ^1111      ^1121      ^1131      ^1141      ^1151      ^1161      ^1171      ^1181      ^1191      

ACAGCGCGGG CTTCTAGCGC TCGGCACCGG CGGGCCAGGC GCGTCCTGCC TTCATTTATC CAGCAGCTTT TCGGAAAATG CATTTGCTGT TCGGAGTTTA 
^1201      ^1211      ^1221      ^1231      ^1241      ^1251      ^1261      ^1271      ^1281      ^1291      

ATCAGAAGAC GATTCCTGCC TCCGTCCCCG GCTCCTTCAT CGTCCCATCT CCCCTGTCTC TCTCCTGGGG AGGCGTGAAG CGGTCCCGTG GATAGAGATT 
^1301      ^1311      ^1321      ^1331      ^1341      ^1351      ^1361      ^1371      ^1381      ^1391      

CATGCCTGTG TCCGCGCGTG TGTGCGCGCG TATAAATTGC CGAGAAGGGG AAAACATCAC AGGACTTCTG CGAATACCGG ACTGAAAATT GTAATTCATC 
^1401      ^1411      ^1421      ^1431      ^1441      ^1451      ^1461      ^1471      ^1481      ^1491      

TGCCGCCGCC GCTGCCTTTT TTTTTTCTCG AGCTCTTGAG ATCTCCGGTT GGGATTCCTG CGGATTGACA TTTCTGTGAA GCAGAAGTCT GGGAATCGAT 
^1501      ^1511      ^1521      ^1531      ^1541      ^1551      ^1561      ^1571      ^1581      ^1591      

CTGGAAATCC TCCTAATTTT TACTCCCTCT CCCCCCGACT CCTGATTCAT TGGGAAGTTT CAAATCAGCT ATAACTGGAG AGTGCTGAAG ATTGATGGGA 
^1601      ^1611      ^1621      ^1631      ^1641      ^1651      ^1661      ^1671      ^1681      ^1691      

TCGTTGCCTT ATGCATTTGT TTTGGTTTTA CAAAAAGGAA ACTTGACAGA GGATCATGCT GTACTTAAAA AATACAAGTA AGTCTCGCAC AGGAAATTGG 
^1701      ^1711      ^1721      ^1731      ^1741      ^1751      ^1761      ^1771      ^1781      ^1791      

TTTAATGTAA CTTTCAATGG AAACCTTTGA GATTTTTTAC TTAAAGTGCA TTCGAGTAAA TTTAATTTCC AGGCAGCTTA ATACATTGTT TTTAGCCGTG 
^1801      ^1811      ^1821      ^1831      ^1841      ^1851      ^1861      ^1871      ^1881      ^1891      

TTACTTGTAG TGTGTATGCC CTGCTTTCAC TCAGTGTGTA CAGGGAAACG CACCTGATTT TTTACTTATT AGTTTGTTTT TTCTTTAACC TTTCAGCATC 
^1901      ^1911      ^1921      ^1931      ^1941      ^1951      ^1961      ^1971      ^1981      ^1991      

ACAGAGGAAG TAGACTGATA TTAACAATAC TTACTAATAA TAACGTGCCT CATGAAATAA AGATCCGAAA GGAATTGGAA TAAAAATTTC CTGCGTCTCA 
^2001      ^2011      ^2021      ^2031      ^2041      ^2051      ^2061      ^2071      ^2081      ^2091      

TGCCAAGAGG GAAACACCAG AATCAAGTGT TCCGCGTGAT TGAAGACACC CCCTCGTCCA AGAATGCAAA GCACATCCAA TAAAATAGCT GGATTATAAC 
^2101      ^2111      ^2121      ^2131      ^2141      ^2151      ^2161      ^2171      ^2181      ^2191      

TCCTCTTCTT TCTCTGGGGG CCGTGGGGTG GGAGCTGGGG CGAGAGGTGC CGTTGGCCCC CGTTGCTTTT CTTCTGGGAA GCATGGGCCC ACAGGACGTC 
^2201      ^2211      ^2221      ^2231      ^2241      ^2251      ^2261      ^2271      ^2281      ^2291      

ATGGAAGACG CCAAAAACAT AAAGAAAGGC CCGGCGCCAT TCTATCCTCT AGAGGATGGA ACCGCTGGAG AGCAACTGCA TAAGGCTATG AAGAGATACG 
^2301      ^2311      ^2321      ^2331      ^2341      ^2351      ^2361      ^2371      ^2381      ^2391      

CCCTGGTTCC TGGAACAATT GCTTTTACAG ATGCACATAT CGAGGTGAAC ATCACGTACG CGGAATACTT CGAAATGTCC GTTCGGTTGG CAGAAGCTAT 
^2401      ^2411      ^2421      ^2431      ^2441      ^2451      ^2461      ^2471      ^2481      ^2491      

GAAACGATAT GGGCTGAATA CAAATCACAG AATCGTCGTA TGCAGTGAAA ACTCTCTTCA ATTCTTTATG CCGGTGTTGG GCGCGTTATT TATCGGAGTT 
^2501      ^2511      ^2521      ^2531      ^2541      ^2551      ^2561      ^2571      ^2581      ^2591      

GCAGTTGCGC CCGCGAACGA CATTTATAAT GAACGTGAAT TGCTCAACAG TATGAACATT TCGCAGCCTA CCGTAGTGTT TGTTTCCAAA AAGGGGTTGC 
^2601      ^2611      ^2621      ^2631      ^2641      ^2651      ^2661      ^2671      ^2681      ^2691      

AAAAAATTTT GAACGTGCAA AAAAAATTAC CAATAATCCA GAAAATTATT ATCATGGATT CTAAAACGGA TTACCAGGGA TTTCAGTCGA TGTACACGTT 
^2701      ^2711      ^2721      ^2731      ^2741      ^2751      ^2761      ^2771      ^2781      ^2791      

CGTCACATCT CATCTACCTC CCGGTTTTAA TGAATACGAT TTTGTACCAG AGTCCTTTGA TCGTGACAAA ACAATTGCAC TGATAATGAA TTCCTCTGGA 
^2801      ^2811      ^2821      ^2831      ^2841      ^2851      ^2861      ^2871      ^2881      ^2891      

TCTACTGGGT TACCTAAGGG TGTGGCCCTT CCGCATAGAA CTGCCTGCGT CAGATTCTCG CATGCCAGAG ATCCTATTTT TGGCAATCAA ATCATTCCGG 
^2901      ^2911      ^2921      ^2931      ^2941      ^2951      ^2961      ^2971      ^2981      ^2991      

ATACTGCGAT TTTAAGTGTT GTTCCATTCC ATCACGGTTT TGGAATGTTT ACTACACTCG GATATTTGAT ATGTGGATTT CGAGTCGTCT TAATGTATAG 
^3001      ^3011      ^3021      ^3031      ^3041      ^3051      ^3061      ^3071      ^3081      ^3091      

ATTTGAAGAA GAGCTGTTTT TACGATCCCT TCAGGATTAC AAAATTCAAA GTGCGTTGCT AGTACCAACC CTATTTTCAT TCTTCGCCAA AAGCACTCTG 
^3101      ^3111      ^3121      ^3131      ^3141      ^3151      ^3161      ^3171      ^3181      ^3191      

ATTGACAAAT ACGATTTATC TAATTTACAC GAAATTGCTT CTGGGGGCGC ACCTCTTTCG AAAGAAGTCG GGGAAGCGGT TGCAAAACGC TTCCATCTTC 
^3201      ^3211      ^3221      ^3231      ^3241      ^3251      ^3261      ^3271      ^3281      ^3291      

CAGGGATACG ACAAGGATAT GGGCTCACTG AGACTACATC AGCTATTCTG ATTACACCCG AGGGGGATGA TAAACCGGGC GCGGTCGGTA AAGTTGTTCC 
^3301      ^3311      ^3321      ^3331      ^3341      ^3351      ^3361      ^3371      ^3381      ^3391      

ATTTTTTGAA GCGAAGGTTG TGGATCTGGA TACCGGGAAA ACGCTGGGCG TTAATCAGAG AGGCGAATTA TGTGTCAGAG GACCTATGAT TATGTCCGGT 
^3401      ^3411      ^3421      ^3431      ^3441      ^3451      ^3461      ^3471      ^3481      ^3491      

TATGTAAACA ATCCGGAAGC GACCAACGCC TTGATTGACA AGGATGGATG GCTACATTCT GGAGACATAG CTTACTGGGA CGAAGACGAA CACTTCTTCA 
^3501      ^3511      ^3521      ^3531      ^3541      ^3551      ^3561      ^3571      ^3581      ^3591      

TAGTTGACCG CTTGAAGTCT TTAATTAAAT ACAAAGGATA TCAGGTGGCC CCCGCTGAAT TGGAATCGAT ATTGTTACAA CACCCCAACA TCTTCGACGC 
^3601      ^3611      ^3621      ^3631      ^3641      ^3651      ^3661      ^3671      ^3681      ^3691      

GGGCGTGGCA GGTCTTCCCG ACGATGACGC CGGTGAACTT CCCGCCGCCG TTGTTGTTTT GGAGCACGGA AAGACGATGA CGGAAAAAGA GATCGTGGAT 
^3701      ^3711      ^3721      ^3731      ^3741      ^3751      ^3761      ^3771      ^3781      ^3791      

TACGTGGCCA GTCAAGTAAC AACCGCGAAA AAGTTGCGCG GAGGAGTTGT GTTTGTGGAC GAAGTACCGA AAGGTCTTAC CGGAAAACTC GACGCAAGAA 
^3801      ^3811      ^3821      ^3831      ^3841      ^3851      ^3861      ^3871      ^3881      ^3891      

AAATCAGAGA GATCCTCATA AAGGCCAAGA AGGGCGGAAA GTCCAAATTG TAAGGATCCG GGCCCTATTC TATAGTGTCA CCTAAATGCT AGAGCTCGCT 
^3901      ^3911      ^3921      ^3931      ^3941      ^3951      ^3961      ^3971      ^3981      ^3991      

GATCAGCCTC GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC 
^4001      ^4011      ^4021      ^4031      ^4041      ^4051      ^4061      ^4071      ^4081      ^4091      

CTAATAAAAT GAGGAAATTG CATCGC
^4101      ^4111      ^4121

Warning: mRNA sequence when devoid of trailing 'A's is still not a substring of the plasmid sequence. Is it because an intron is spliced out? Stay calm then. :-)

Credibility of mRNA sequence:

reverse_engineered_sequence_and_should_match_experiment_except_3UTR

The name of the plasmid:
pRL-BCL2-FL The name of the promoter used to express this mRNA:
CMV

The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):

yes

The in vivo produced heterogeneous transcripts occur due to alternative splicing:

yes

A promoter reported in cDNA corresponding to IRES sequence:

yes

Summary of possible issues when IRES cDNA is experimentally transcribed in vivo:

Summary of experiments studying integrity of the in vivo transcripts in a particular host:

Integrity (uniformity) of mRNA tested using Northern-blot:
not_tested

Integrity (uniformity) of mRNA tested using RNase protection:
not_tested

Integrity (uniformity) of mRNA tested using 5'-RACE:
not_tested

Integrity (uniformity) of mRNA tested using primer extension :
not_tested

Integrity (uniformity) of mRNA tested using RT-PCR:
not_tested

Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR):
not_tested

Integrity (uniformity) of mRNA tested using RNAi:
not_tested

Integrity (uniformity) of mRNA tested using S1 nuclease mapping:
not_tested

Cryptic promoter presence was confirmed by expression from a promoter-less plasmid:
weak_promoter_confirmed

Cryptic promoter presence was confirmed in an experimental setup involving inducible promoter:
not_tested

Integrity (uniformity) of mRNA molecules or possible promoter presence expressed in vivo was tested using another method, please specify in Remarks:
not_tested

The organism used:

Homo sapiens HEK 293T/17 (ATCC CRL-11268)

The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
BCL2

The origin of IRES in the plasmid:

nuclear

The donor organism of the IRES segment:

Homo sapiens HeLa (ATCC CCL-2)

The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:

ATTAAACCTC GATCTTCAAT ATTGGCCATT AGCCATATTA TTCATTGGTT ATATAGCATA AATCAATATT GGCTATTGGC CATTGCATAC GTTGTATCTA 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

TATCATAATA TGTACATTTA TATTGGCTCA TGTCCAATAT GACCGCCATG TTGGCATTGA TTATTGACTA GTTATTAATA GTAATCAATT ACGGGGTCAT 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

TAGTTCATAG CCCATATATG GAGTTCCGCG TTACATAACT TACGGTAAAT GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

CATATGCCAA GTCCGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACA TGACCTTACG GGACTTTCCT ACTTGGCAGT 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG TACACCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAT AACCCCGCCC CGTTGACGCA AATGGGCGGT 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTCGTT TAGTGAACCG TCAGATCACT AGAAGCTTTA TTGCGGTAGT TTATCACAGT TAAATTGCTA 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

ACGCAGTCAG TGCTTCTGAC ACAACAGTCT CGAACTTAAG CTGCAGAAGT TGGTCGTGAG GCACTGGGCA GGTAAGTATC AAGGTTACAA GACAGGTTTA 
^801       ^811       ^821       ^831       ^841       ^851       ^861       ^871       ^881       ^891       

AGGAGACCAA TAGAAACTGG GCTTGTCGAG ACAGAGAAGA CTCTTGCGTT TCTGATAGGC ACCTATTGGT CTTACTGACA TCCACTTTGC CTTTCTCTCC 
^901       ^911       ^921       ^931       ^941       ^951       ^961       ^971       ^981       ^991       

ACAGGTGTCC ACTCCCAGTT CAATTACAGC TCTTAAGGCT AGAGTACTTA ATACGACTCA CTATAGGCTA GCCACCATGA CTTCGAAAGT TTATGATCCA 
^1001      ^1011      ^1021      ^1031      ^1041      ^1051      ^1061      ^1071      ^1081      ^1091      

GAACAAAGGA AACGGATGAT AACTGGTCCG CAGTGGTGGG CCAGATGTAA ACAAATGAAT GTTCTTGATT CATTTATTAA TTATTATGAT TCAGAAAAAC 
^1101      ^1111      ^1121      ^1131      ^1141      ^1151      ^1161      ^1171      ^1181      ^1191      

ATGCAGAAAA TGCTGTTATT TTTTTACATG GTAACGCGGC CTCTTCTTAT TTATGGCGAC ATGTTGTGCC ACATATTGAG CCAGTAGCGC GGTGTATTAT 
^1201      ^1211      ^1221      ^1231      ^1241      ^1251      ^1261      ^1271      ^1281      ^1291      

ACCAGACCTT ATTGGTATGG GCAAATCAGG CAAATCTGGT AATGGTTCTT ATAGGTTACT TGATCATTAC AAATATCTTA CTGCATGGTT TGAACTTCTT 
^1301      ^1311      ^1321      ^1331      ^1341      ^1351      ^1361      ^1371      ^1381      ^1391      

AATTTACCAA AGAAGATCAT TTTTGTCGGC CATGATTGGG GTGCTTGTTT GGCATTTCAT TATAGCTATG AGCATCAAGA TAAGATCAAA GCAATAGTTC 
^1401      ^1411      ^1421      ^1431      ^1441      ^1451      ^1461      ^1471      ^1481      ^1491      

ACGCTGAAAG TGTAGTAGAT GTGATTGAAT CATGGGATGA ATGGCCTGAT ATTGAAGAAG ATATTGCGTT GATCAAATCT GAAGAAGGAG AAAAAATGGT 
^1501      ^1511      ^1521      ^1531      ^1541      ^1551      ^1561      ^1571      ^1581      ^1591      

TTTGGAGAAT AACTTCTTCG TGGAAACCAT GTTGCCATCA AAAATCATGA GAAAGTTAGA ACCAGAAGAA TTTGCAGCAT ATCTTGAACC ATTCAAAGAG 
^1601      ^1611      ^1621      ^1631      ^1641      ^1651      ^1661      ^1671      ^1681      ^1691      

AAAGGTGAAG TTCGTCGTCC AACATTATCA TGGCCTCGTG AAATCCCGTT AGTAAAAGGT GGTAAACCTG ACGTTGTACA AATTGTTAGG AATTATAATG 
^1701      ^1711      ^1721      ^1731      ^1741      ^1751      ^1761      ^1771      ^1781      ^1791      

CTTATCTACG TGCAAGTGAT GATTTACCAA AAATGTTTAT TGAATCGGAC CCAGGATTCT TTTCCAATGC TATTGTTGAA GGTGCCAAGA AGTTTCCTAA 
^1801      ^1811      ^1821      ^1831      ^1841      ^1851      ^1861      ^1871      ^1881      ^1891      

TACTGAATTT GTCAAAGTAA AAGGTCTTCA TTTTTCGCAA GAAGATGCAC CTGATGAAAT GGGAAAATAT ATCAAATCGT TCGTTGAGCG AGTTCTCAAA 
^1901      ^1911      ^1921      ^1931      ^1941      ^1951      ^1961      ^1971      ^1981      ^1991      

AATGAACAAT AATTCTAGAG CGGCCGCAAA AAATAAAACC CTCCCCCACC ACCTCCTTCT CCCCACCCCT CGCCGCACCA CACACAGCGC GGGCTTCTAG 
^2001      ^2011      ^2021      ^2031      ^2041      ^2051      ^2061      ^2071      ^2081      ^2091      

CGCTCGGCAC CGGCGGGCCA GGCGCGTCCT GCCTTCATTT ATCCAGCAGC TTTTCGGAAA ATGCATTTGC TGTTCGGAGT TTAATCAGAA GACGATTCCT 
^2101      ^2111      ^2121      ^2131      ^2141      ^2151      ^2161      ^2171      ^2181      ^2191      

GCCTCCGTCC CCGGCTCCTT CATCGTCCCA TCTCCCCTGT CTCTCTCCTG GGGAGGCGTG AAGCGGTCCC GTGGATAGAG ATTCATGCCT GTGTCCGCGC 
^2201      ^2211      ^2221      ^2231      ^2241      ^2251      ^2261      ^2271      ^2281      ^2291      

GTGTGTGCGC GCGTATAAAT TGCCGAGAAG GGGAAAACAT CACAGGACTT CTGCGAATAC CGGACTGAAA ATTGTAATTC ATCTGCCGCC GCCGCTGCCT 
^2301      ^2311      ^2321      ^2331      ^2341      ^2351      ^2361      ^2371      ^2381      ^2391      

TTTTTTTTTC TCGAGCTCTT GAGATCTCCG GTTGGGATTC CTGCGGATTG ACATTTCTGT GAAGCAGAAG TCTGGGAATC GATCTGGAAA TCCTCCTAAT 
^2401      ^2411      ^2421      ^2431      ^2441      ^2451      ^2461      ^2471      ^2481      ^2491      

TTTTACTCCC TCTCCCCCCG ACTCCTGATT CATTGGGAAG TTTCAAATCA GCTATAACTG GAGAGTGCTG AAGATTGATG GGATCGTTGC CTTATGCATT 
^2501      ^2511      ^2521      ^2531      ^2541      ^2551      ^2561      ^2571      ^2581      ^2591      

TGTTTTGGTT TTACAAAAAG GAAACTTGAC AGAGGATCAT GCTGTACTTA AAAAATACAA GTAAGTCTCG CACAGGAAAT TGGTTTAATG TAACTTTCAA 
^2601      ^2611      ^2621      ^2631      ^2641      ^2651      ^2661      ^2671      ^2681      ^2691      

TGGAAACCTT TGAGATTTTT TACTTAAAGT GCATTCGAGT AAATTTAATT TCCAGGCAGC TTAATACATT GTTTTTAGCC GTGTTACTTG TAGTGTGTAT 
^2701      ^2711      ^2721      ^2731      ^2741      ^2751      ^2761      ^2771      ^2781      ^2791      

GCCCTGCTTT CACTCAGTGT GTACAGGGAA ACGCACCTGA TTTTTTACTT ATTAGTTTGT TTTTTCTTTA ACCTTTCAGC ATCACAGAGG AAGTAGACTG 
^2801      ^2811      ^2821      ^2831      ^2841      ^2851      ^2861      ^2871      ^2881      ^2891      

ATATTAACAA TACTTACTAA TAATAACGTG CCTCATGAAA TAAAGATCCG AAAGGAATTG GAATAAAAAT TTCCTGCGTC TCATGCCAAG AGGGAAACAC 
^2901      ^2911      ^2921      ^2931      ^2941      ^2951      ^2961      ^2971      ^2981      ^2991      

CAGAATCAAG TGTTCCGCGT GATTGAAGAC ACCCCCTCGT CCAAGAATGC AAAGCACATC CAATAAAATA GCTGGATTAT AACTCCTCTT CTTTCTCTGG 
^3001      ^3011      ^3021      ^3031      ^3041      ^3051      ^3061      ^3071      ^3081      ^3091      

GGGCCGTGGG GTGGGAGCTG GGGCGAGAGG TGCCGTTGGC CCCCGTTGCT TTTCTTCTGG GAAGCATGGG CCCACAGGAC GTCATGGAAG ACGCCAAAAA 
^3101      ^3111      ^3121      ^3131      ^3141      ^3151      ^3161      ^3171      ^3181      ^3191      

CATAAAGAAA GGCCCGGCGC CATTCTATCC TCTAGAGGAT GGAACCGCTG GAGAGCAACT GCATAAGGCT ATGAAGAGAT ACGCCCTGGT TCCTGGAACA 
^3201      ^3211      ^3221      ^3231      ^3241      ^3251      ^3261      ^3271      ^3281      ^3291      

ATTGCTTTTA CAGATGCACA TATCGAGGTG AACATCACGT ACGCGGAATA CTTCGAAATG TCCGTTCGGT TGGCAGAAGC TATGAAACGA TATGGGCTGA 
^3301      ^3311      ^3321      ^3331      ^3341      ^3351      ^3361      ^3371      ^3381      ^3391      

ATACAAATCA CAGAATCGTC GTATGCAGTG AAAACTCTCT TCAATTCTTT ATGCCGGTGT TGGGCGCGTT ATTTATCGGA GTTGCAGTTG CGCCCGCGAA 
^3401      ^3411      ^3421      ^3431      ^3441      ^3451      ^3461      ^3471      ^3481      ^3491      

CGACATTTAT AATGAACGTG AATTGCTCAA CAGTATGAAC ATTTCGCAGC CTACCGTAGT GTTTGTTTCC AAAAAGGGGT TGCAAAAAAT TTTGAACGTG 
^3501      ^3511      ^3521      ^3531      ^3541      ^3551      ^3561      ^3571      ^3581      ^3591      

CAAAAAAAAT TACCAATAAT CCAGAAAATT ATTATCATGG ATTCTAAAAC GGATTACCAG GGATTTCAGT CGATGTACAC GTTCGTCACA TCTCATCTAC 
^3601      ^3611      ^3621      ^3631      ^3641      ^3651      ^3661      ^3671      ^3681      ^3691      

CTCCCGGTTT TAATGAATAC GATTTTGTAC CAGAGTCCTT TGATCGTGAC AAAACAATTG CACTGATAAT GAATTCCTCT GGATCTACTG GGTTACCTAA 
^3701      ^3711      ^3721      ^3731      ^3741      ^3751      ^3761      ^3771      ^3781      ^3791      

GGGTGTGGCC CTTCCGCATA GAACTGCCTG CGTCAGATTC TCGCATGCCA GAGATCCTAT TTTTGGCAAT CAAATCATTC CGGATACTGC GATTTTAAGT 
^3801      ^3811      ^3821      ^3831      ^3841      ^3851      ^3861      ^3871      ^3881      ^3891      

GTTGTTCCAT TCCATCACGG TTTTGGAATG TTTACTACAC TCGGATATTT GATATGTGGA TTTCGAGTCG TCTTAATGTA TAGATTTGAA GAAGAGCTGT 
^3901      ^3911      ^3921      ^3931      ^3941      ^3951      ^3961      ^3971      ^3981      ^3991      

TTTTACGATC CCTTCAGGAT TACAAAATTC AAAGTGCGTT GCTAGTACCA ACCCTATTTT CATTCTTCGC CAAAAGCACT CTGATTGACA AATACGATTT 
^4001      ^4011      ^4021      ^4031      ^4041      ^4051      ^4061      ^4071      ^4081      ^4091      

ATCTAATTTA CACGAAATTG CTTCTGGGGG CGCACCTCTT TCGAAAGAAG TCGGGGAAGC GGTTGCAAAA CGCTTCCATC TTCCAGGGAT ACGACAAGGA 
^4101      ^4111      ^4121      ^4131      ^4141      ^4151      ^4161      ^4171      ^4181      ^4191      

TATGGGCTCA CTGAGACTAC ATCAGCTATT CTGATTACAC CCGAGGGGGA TGATAAACCG GGCGCGGTCG GTAAAGTTGT TCCATTTTTT GAAGCGAAGG 
^4201      ^4211      ^4221      ^4231      ^4241      ^4251      ^4261      ^4271      ^4281      ^4291      

TTGTGGATCT GGATACCGGG AAAACGCTGG GCGTTAATCA GAGAGGCGAA TTATGTGTCA GAGGACCTAT GATTATGTCC GGTTATGTAA ACAATCCGGA 
^4301      ^4311      ^4321      ^4331      ^4341      ^4351      ^4361      ^4371      ^4381      ^4391      

AGCGACCAAC GCCTTGATTG ACAAGGATGG ATGGCTACAT TCTGGAGACA TAGCTTACTG GGACGAAGAC GAACACTTCT TCATAGTTGA CCGCTTGAAG 
^4401      ^4411      ^4421      ^4431      ^4441      ^4451      ^4461      ^4471      ^4481      ^4491      

TCTTTAATTA AATACAAAGG ATATCAGGTG GCCCCCGCTG AATTGGAATC GATATTGTTA CAACACCCCA ACATCTTCGA CGCGGGCGTG GCAGGTCTTC 
^4501      ^4511      ^4521      ^4531      ^4541      ^4551      ^4561      ^4571      ^4581      ^4591      

CCGACGATGA CGCCGGTGAA CTTCCCGCCG CCGTTGTTGT TTTGGAGCAC GGAAAGACGA TGACGGAAAA AGAGATCGTG GATTACGTGG CCAGTCAAGT 
^4601      ^4611      ^4621      ^4631      ^4641      ^4651      ^4661      ^4671      ^4681      ^4691      

AACAACCGCG AAAAAGTTGC GCGGAGGAGT TGTGTTTGTG GACGAAGTAC CGAAAGGTCT TACCGGAAAA CTCGACGCAA GAAAAATCAG AGAGATCCTC 
^4701      ^4711      ^4721      ^4731      ^4741      ^4751      ^4761      ^4771      ^4781      ^4791      

ATAAAGGCCA AGAAGGGCGG AAAGTCCAAA TTGTAAGGAT CCGGGCCCTA TTCTATAGTG TCACCTAAAT GCTAGAGCTC GCTGATCAGC CTCGACTGTG 
^4801      ^4811      ^4821      ^4831      ^4841      ^4851      ^4861      ^4871      ^4881      ^4891      

CCTTCTAGTT GCCAGCCATC TGTTGTTTGC CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT TTCCTAATAA AATGAGGAAA 
^4901      ^4911      ^4921      ^4931      ^4941      ^4951      ^4961      ^4971      ^4981      ^4991      

TTGCATCGCA TTGTCTGAGT AGGTGTCATT CTATTCTGGG GGGTGGGGTG GGGCAGGACA GCAAGGGGGA GGATTGGGAA GACAATAGCA GGCATGCTGG 
^5001      ^5011      ^5021      ^5031      ^5041      ^5051      ^5061      ^5071      ^5081      ^5091      

GGATGCGGTG GGCTCTATGG CTTCTGAGGC GGAAAGAACC AGCTGGGGCT CGAGGGGGGA TCCCCACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGT 
^5101      ^5111      ^5121      ^5131      ^5141      ^5151      ^5161      ^5171      ^5181      ^5191      

GTGGTGGTTA CGCGCAGCGT GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC GCTTTCTTCC CTTCCTTTCT CGCCACGTTC GCCGGCTTTC 
^5201      ^5211      ^5221      ^5231      ^5241      ^5251      ^5261      ^5271      ^5281      ^5291      

CCCGTCAAGC TCTAAATCGG GGCATCCCTT TAGGGTTCCG ATTTAGTGCT TTACGGCACC TCGACCCCAA AAAACTTGAT TAGGGTGATG GTTCACGTAG 
^5301      ^5311      ^5321      ^5331      ^5341      ^5351      ^5361      ^5371      ^5381      ^5391      

TGGGCCATCG CCCTGATAGA CGGTTTTTCG CCCTTTGACG TTGGAGTCCA CGTTCTTTAA TAGTGGACTC TTGTTCCAAA CTGGAACAAC ACTCAACCCT 
^5401      ^5411      ^5421      ^5431      ^5441      ^5451      ^5461      ^5471      ^5481      ^5491      

ATCTCGGTCT ATTCTTTTGA TTTATAAGGG ATTTTGGGGA TTTCGGCCTA TTGGTTAAAA AATGAGCTGA TTTAACAAAA ATTTAACGCG AATTTTAACA 
^5501      ^5511      ^5521      ^5531      ^5541      ^5551      ^5561      ^5571      ^5581      ^5591      

AAATATTAAC GTTTACAATT TAAATATTTG CTTATACAAT CTTCCTGTTT TTGGGGCTTT TCTGATTATC AACCGGGGTG GGTACCGAGC TCGAATTCTG 
^5601      ^5611      ^5621      ^5631      ^5641      ^5651      ^5661      ^5671      ^5681      ^5691      

TGGAATGTGT GTCAGTTAGG GTGTGGAAAG TCCCCAGGCT CCCCAGGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CAGGTGTGGA 
^5701      ^5711      ^5721      ^5731      ^5741      ^5751      ^5761      ^5771      ^5781      ^5791      

AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC CCGCCCCTAA 
^5801      ^5811      ^5821      ^5831      ^5841      ^5851      ^5861      ^5871      ^5881      ^5891      

CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT ATTTATGCAG AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA 
^5901      ^5911      ^5921      ^5931      ^5941      ^5951      ^5961      ^5971      ^5981      ^5991      

GTGAGGAGGC TTTTTTGGAG GCCTAGGCTT TTGCAAAAAG CTCCCGGGAG CTTGGATATC CATTTTCGGA TCTGATCAAG AGACAGGATG AGGATCGTTT 
^6001      ^6011      ^6021      ^6031      ^6041      ^6051      ^6061      ^6071      ^6081      ^6091      

CGCATGATTG AACAAGATGG ATTGCACGCA GGTTCTCCGG CCGCTTGGGT GGAGAGGCTA TTCGGCTATG ACTGGGCACA ACAGACAATC GGCTGCTCTG 
^6101      ^6111      ^6121      ^6131      ^6141      ^6151      ^6161      ^6171      ^6181      ^6191      

ATGCCGCCGT GTTCCGGCTG TCAGCGCAGG GGCGCCCGGT TCTTTTTGTC AAGACCGACC TGTCCGGTGC CCTGAATGAA CTGCAGGACG AGGCAGCGCG 
^6201      ^6211      ^6221      ^6231      ^6241      ^6251      ^6261      ^6271      ^6281      ^6291      

GCTATCGTGG CTGGCCACGA CGGGCGTTCC TTGCGCAGCT GTGCTCGACG TTGTCACTGA AGCGGGAAGG GACTGGCTGC TATTGGGCGA AGTGCCGGGG 
^6301      ^6311      ^6321      ^6331      ^6341      ^6351      ^6361      ^6371      ^6381      ^6391      

CAGGATCTCC TGTCATCTCA CCTTGCTCCT GCCGAGAAAG TATCCATCAT GGCTGATGCA ATGCGGCGGC TGCATACGCT TGATCCGGCT ACCTGCCCAT 
^6401      ^6411      ^6421      ^6431      ^6441      ^6451      ^6461      ^6471      ^6481      ^6491      

TCGACCACCA AGCGAAACAT CGCATCGAGC GAGCACGTAC TCGGATGGAA GCCGGTCTTG TCGATCAGGA TGATCTGGAC GAAGAGCATC AGGGGCTCGC 
^6501      ^6511      ^6521      ^6531      ^6541      ^6551      ^6561      ^6571      ^6581      ^6591      

GCCAGCCGAA CTGTTCGCCA GGCTCAAGGC GCGCATGCCC GACGGCGAGG ATCTCGTCGT GACCCATGGC GATGCCTGCT TGCCGAATAT CATGGTGGAA 
^6601      ^6611      ^6621      ^6631      ^6641      ^6651      ^6661      ^6671      ^6681      ^6691      

AATGGCCGCT TTTCTGGATT CATCGACTGT GGCCGGCTGG GTGTGGCGGA CCGCTATCAG GACATAGCGT TGGCTACCCG TGATATTGCT GAAGAGCTTG 
^6701      ^6711      ^6721      ^6731      ^6741      ^6751      ^6761      ^6771      ^6781      ^6791      

GCGGCGAATG GGCTGACCGC TTCCTCGTGC TTTACGGTAT CGCCGCTCCC GATTCGCAGC GCATCGCCTT CTATCGCCTT CTTGACGAGT TCTTCTGAGC 
^6801      ^6811      ^6821      ^6831      ^6841      ^6851      ^6861      ^6871      ^6881      ^6891      

GGGACTCTGG GGTTCGAAAT GACCGACCAA GCGACGCCCA ACCTGCCATC ACGAGATTTC GATTCCACCG CCGCCTTCTA TGAAAGGTTG GGCTTCGGAA 
^6901      ^6911      ^6921      ^6931      ^6941      ^6951      ^6961      ^6971      ^6981      ^6991      

TCGTTTTCCG GGACGCCGGC TGGATGATCC TCCAGCGCGG GGATCTCATG CTGGAGTTCT TCGCCCACCC CAACTTGTTT ATTGCAGCTT ATAATGGTTA 
^7001      ^7011      ^7021      ^7031      ^7041      ^7051      ^7061      ^7071      ^7081      ^7091      

CAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCATTCTAG TTGTGGTTTG TCCAAACTCA TCAATGTATC TTATCATGTC 
^7101      ^7111      ^7121      ^7131      ^7141      ^7151      ^7161      ^7171      ^7181      ^7191      

TGGATCCCGT CGACCTCGAG AGCTTGGCGT AATCATGGTC ATAGCTGTTT CCTGTGTGAA ATTGTTATCC GCTCACAATT CCACACAACA TACGAGCCGG 
^7201      ^7211      ^7221      ^7231      ^7241      ^7251      ^7261      ^7271      ^7281      ^7291      

AAGCATAAAG TGTAAAGCCT GGGGTGCCTA ATGAGTGAGC TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC 
^7301      ^7311      ^7321      ^7331      ^7341      ^7351      ^7361      ^7371      ^7381      ^7391      

CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC 
^7401      ^7411      ^7421      ^7431      ^7441      ^7451      ^7461      ^7471      ^7481      ^7491      

GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA 
^7501      ^7511      ^7521      ^7531      ^7541      ^7551      ^7561      ^7571      ^7581      ^7591      

AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG 
^7601      ^7611      ^7621      ^7631      ^7641      ^7651      ^7661      ^7671      ^7681      ^7691      

CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG 
^7701      ^7711      ^7721      ^7731      ^7741      ^7751      ^7761      ^7771      ^7781      ^7791      

CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCAATGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA 
^7801      ^7811      ^7821      ^7831      ^7841      ^7851      ^7861      ^7871      ^7881      ^7891      

ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT 
^7901      ^7911      ^7921      ^7931      ^7941      ^7951      ^7961      ^7971      ^7981      ^7991      

AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG 
^8001      ^8011      ^8021      ^8031      ^8041      ^8051      ^8061      ^8071      ^8081      ^8091      

CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA 
^8101      ^8111      ^8121      ^8131      ^8141      ^8151      ^8161      ^8171      ^8181      ^8191      

GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC 
^8201      ^8211      ^8221      ^8231      ^8241      ^8251      ^8261      ^8271      ^8281      ^8291      

ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT 
^8301      ^8311      ^8321      ^8331      ^8341      ^8351      ^8361      ^8371      ^8381      ^8391      

ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA 
^8401      ^8411      ^8421      ^8431      ^8441      ^8451      ^8461      ^8471      ^8481      ^8491      

GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG 
^8501      ^8511      ^8521      ^8531      ^8541      ^8551      ^8561      ^8571      ^8581      ^8591      

CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG 
^8601      ^8611      ^8621      ^8631      ^8641      ^8651      ^8661      ^8671      ^8681      ^8691      

TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC 
^8701      ^8711      ^8721      ^8731      ^8741      ^8751      ^8761      ^8771      ^8781      ^8791      

CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT 
^8801      ^8811      ^8821      ^8831      ^8841      ^8851      ^8861      ^8871      ^8881      ^8891      

AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT 
^8901      ^8911      ^8921      ^8931      ^8941      ^8951      ^8961      ^8971      ^8981      ^8991      

CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT 
^9001      ^9011      ^9021      ^9031      ^9041      ^9051      ^9061      ^9071      ^9081      ^9091      

CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA 
^9101      ^9111      ^9121      ^9131      ^9141      ^9151      ^9161      ^9171      ^9181      ^9191      

GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT 
^9201      ^9211      ^9221      ^9231      ^9241      ^9251      ^9261      ^9271      ^9281      ^9291      

ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCGACGG 
^9301      ^9311      ^9321      ^9331      ^9341      ^9351      ^9361      ^9371      ^9381      ^9391      

ATCGGGA
^9401

GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:

The total number of notable open-reading frames (ORFs):

2

Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:

ORF

ORF position:

1

Version: `0`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš

The abbreviated name of this ORF/gene:
RLuc

The description of the protein encoded in this ORF:
Renilla luciferase

The translational frameshift (ribosome slippage) involved:
0 The ribosome read-through involved:
no

The alternative forms of this protein occur by the alternative initiation of translation:

not tested

The ORF absolute position (the base range includes START and STOP codons or their equivalents):

190-1125

ORF

ORF position:

2

Version: `1`	Last change: `2006-10-30 00:00:00`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš

The abbreviated name of this ORF/gene:
FLuc-fusion

The description of the protein encoded in this ORF:
Firefly luciferase fused N-terminally with 6 extra aminoacid residues (including the initiator ATG) from BCL2 protein.

The translational frameshift (ribosome slippage) involved:
0 The ribosome read-through involved:
no

The alternative forms of this protein occur by the alternative initiation of translation:

not tested

The ORF absolute position (the base range includes START and STOP codons or their equivalents):

2279-3949

Remarks:

Negligible promoter activity attributable to BCL2 IRES was shown in Fig. 3A using promoter-less plasmid.
Alternatively spliced transcripts from this plasmid were confirmed in Fig. 3C.

Citations:

Sherrill K. W., Byrd M. P., Van Eden M. E., Lloyd R. E. (2004) BCL-2 translation is mediated via internal ribosome entry during cell stress. J. Biol. Chem. 279(28):29066-29074

IRESs:

IRES:

Version: `4`	Last change: `2007-01-23 00:00:00`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš
The IRES name: `BCL2`	The functional status of IRES: `functional`

The IRES absolute position (the range includes START and STOP codons or their equivalents):

1141-2278

How IRES boundaries were determined:
experimentally_determined

5'-end of IRES relative to last base of the STOP codon of the upstream ORF: `16`	3'-end of IRES relative to last base of the STOP codon of the upstream ORF: `1153`
5'-end of IRES relative to first base of the START codon of the downstream ORF: `-1138`	3'-end of IRES relative to first base of the START codon of the downstream ORF: `-1`

The sequence of IRES region aligned to its secondary structure (if available):

CCGCAAAAAA TAAAACCCTC CCCCACCACC TCCTTCTCCC CACCCCTCGC CGCACCACAC ACAGCGCGGG CTTCTAGCGC TCGGCACCGG CGGGCCAGGC 
^1141      ^1151      ^1161      ^1171      ^1181      ^1191      ^1201      ^1211      ^1221      ^1231      

GCGTCCTGCC TTCATTTATC CAGCAGCTTT TCGGAAAATG CATTTGCTGT TCGGAGTTTA ATCAGAAGAC GATTCCTGCC TCCGTCCCCG GCTCCTTCAT 
^1241      ^1251      ^1261      ^1271      ^1281      ^1291      ^1301      ^1311      ^1321      ^1331      

CGTCCCATCT CCCCTGTCTC TCTCCTGGGG AGGCGTGAAG CGGTCCCGTG GATAGAGATT CATGCCTGTG TCCGCGCGTG TGTGCGCGCG TATAAATTGC 
^1341      ^1351      ^1361      ^1371      ^1381      ^1391      ^1401      ^1411      ^1421      ^1431      

CGAGAAGGGG AAAACATCAC AGGACTTCTG CGAATACCGG ACTGAAAATT GTAATTCATC TGCCGCCGCC GCTGCCTTTT TTTTTTCTCG AGCTCTTGAG 
^1441      ^1451      ^1461      ^1471      ^1481      ^1491      ^1501      ^1511      ^1521      ^1531      

ATCTCCGGTT GGGATTCCTG CGGATTGACA TTTCTGTGAA GCAGAAGTCT GGGAATCGAT CTGGAAATCC TCCTAATTTT TACTCCCTCT CCCCCCGACT 
^1541      ^1551      ^1561      ^1571      ^1581      ^1591      ^1601      ^1611      ^1621      ^1631      

CCTGATTCAT TGGGAAGTTT CAAATCAGCT ATAACTGGAG AGTGCTGAAG ATTGATGGGA TCGTTGCCTT ATGCATTTGT TTTGGTTTTA CAAAAAGGAA 
^1641      ^1651      ^1661      ^1671      ^1681      ^1691      ^1701      ^1711      ^1721      ^1731      

ACTTGACAGA GGATCATGCT GTACTTAAAA AATACAAGTA AGTCTCGCAC AGGAAATTGG TTTAATGTAA CTTTCAATGG AAACCTTTGA GATTTTTTAC 
^1741      ^1751      ^1761      ^1771      ^1781      ^1791      ^1801      ^1811      ^1821      ^1831      

TTAAAGTGCA TTCGAGTAAA TTTAATTTCC AGGCAGCTTA ATACATTGTT TTTAGCCGTG TTACTTGTAG TGTGTATGCC CTGCTTTCAC TCAGTGTGTA 
^1841      ^1851      ^1861      ^1871      ^1881      ^1891      ^1901      ^1911      ^1921      ^1931      

CAGGGAAACG CACCTGATTT TTTACTTATT AGTTTGTTTT TTCTTTAACC TTTCAGCATC ACAGAGGAAG TAGACTGATA TTAACAATAC TTACTAATAA 
^1941      ^1951      ^1961      ^1971      ^1981      ^1991      ^2001      ^2011      ^2021      ^2031      

TAACGTGCCT CATGAAATAA AGATCCGAAA GGAATTGGAA TAAAAATTTC CTGCGTCTCA TGCCAAGAGG GAAACACCAG AATCAAGTGT TCCGCGTGAT 
^2041      ^2051      ^2061      ^2071      ^2081      ^2091      ^2101      ^2111      ^2121      ^2131      

TGAAGACACC CCCTCGTCCA AGAATGCAAA GCACATCCAA TAAAATAGCT GGATTATAAC TCCTCTTCTT TCTCTGGGGG CCGTGGGGTG GGAGCTGGGG 
^2141      ^2151      ^2161      ^2171      ^2181      ^2191      ^2201      ^2211      ^2221      ^2231      

CGAGAGGTGC CGTTGGCCCC CGTTGCTTTT CTTCTGGG
^2241      ^2251      ^2261      ^2271

The translation experiments:

Translation results:

IRESite Translation Id: 96

Version: `4`	Last change: `2006-10-30 00:00:00`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš

The translation method used to study IRES function:
in vivo

The organism used for translation:

Homo sapiens HEK 293T/17 (ATCC CRL-11268)

The temperature (in degrees of Celsia):
37

The relative translation efficiency in % of this IRES:

140.000

Name of IRES used as the positive control:

HCV_type_1b

Name of the plasmid used as the positive control. `pRL-HCV-FL`	Name of the plasmid used as the negative control. `pRL-FL`
`IRESite Id` of the plasmid used as positive control. `131`	`IRESite Id` of the plasmid used as negative control. `119`
The relative translation efficiency in % of the positive control: `100.000`	The relative translation efficiency in % of the negative control: `2.000`
The size (length) of intercistronic region in the positive control: `485`	The size (length) of intercistronic region in the negative control: `28`

The effect of 5'-cap analogs on translation:
not tested

Rapamycin affects translation:
not tested

Type of RNA subject to translation:

endogenous_nuclear_RNA_Pol_II_transcript

Remarks:

The results from Fig. 3A do not form the supporting evidence for existence of IRES in BCL2 (the direct mRNA
transfection is the key experiment, see IRESite Id: 114 namely its subsection IRESite Translation Id: 97).
They only demonstrate how the luciferase values obtained are biased by presence of the cryptic promoter in HCV
IRES and by the promoter in BCL2 and by aberrant splicing from pRL-BCL2-FL plasmid (where splice acceptor site
was uncovered in BCL2 IRES and which causes alternatively splices with the chimeric intron contained in the
plasmid). Those alternative transcripts are functionally monocistronic (Fig. 3B, C).

Citations:

Sherrill K. W., Byrd M. P., Van Eden M. E., Lloyd R. E. (2004) BCL-2 translation is mediated via internal ribosome entry during cell stress. J. Biol. Chem. 279(28):29066-29074

Last change to the database: 2019-03-18 09:32:49 GMT+1