IRESite_Id:605 characteristics of crTMV_IREScp IRES studied in H-CP-IREScp-GUS plasmid

IRESite: The database of experimentally verified IRES structures

The nucleic acid data:

IRESite Id: `605`	Version: `3`
Originaly submitted by: Martin Mokrejš	Submission date: `2009-08-23 18:08:46`
Reviewed by: Martin Mokrejš	Last change: `2009-08-26 13:44:05`

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:
both_UTRs_incomplete

The mRNA/+RNA description:

Part of the T7-derived transcript studied in in vitro assays. The 5'-UTR could have been longer but do not
know the necessary details. The intercistronic region contains IRES sequence of crTMV virus (bp -149 to -1
relative to coat protein ORF).

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

gggcgaattg ggtaccgggc cccccctcga ggtcgacggt atcgataccg tcgacctcga gggggggccc ggtaccATGT CTTACAACAT TACAAACCCG 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

AATCAGTACC AGTATTTCGC AGCGGTGTGG GCAGAGCCCA TTCCGATGCT TAACCAGTGC ATATCGGCAT TGTCACAATC GTACCAAACA CAAGCTGCGA 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

GAGACACTGT TAGACAGCAA TTCTCAAACT TGTTGAGTGC GGTTGTGGCT CCGAGCCAGC GGTTCCCAGA AACAGGGTCC CGGGTGTATG TTAACTCGGC 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

TGTTATAAAG CCGTTGTACG AGGCTCTTAT GAAGTCCTTT GATACTAGAA ATAGGATCAT AGAGACGGAA GAGGAGTCAC GCCCGTCAGC TTCCGAAGTA 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

CGTAACGCGA CACAACGTGT TGATGATGCG ACCGTTTCCA TTAGAAGTCA AATTCAGCTG TTGCTGAGCG AGCTTTCCAG TGGACATGGT TATATGAACA 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

GGGCAGAGTT CGAGGCTTTG GTGCCATGGA CTACTGCTGC TGCTACTTAG aagcttatcg atttcgaacc cggggtaccg aattcgtcga ttcggttgca 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

gcatttaaag cggttgacaa ctttaaaaga aggaaaaaga aggttgaaga aaagggtgta gtaagtaagt ataagtacag accggagaag tacgccggtc 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

ctgattcgtt taatttgaaa gaagaaaccA TGGTCCGTCC TGTAGAAACC CCAACCCGTG AAATCAAAAA ACTCGACGGC CTGTGGGCAT TCAGTCTGGA 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

TCGCGAAAAC TGTGGAATTG ATCAGCGTTG GTGGGAAAGC GCGTTACAAG AAAGCCGGGC AATTGCTGTG CCAGGCAGTT TTAACGATCA GTTCGCCGAT 
^801       ^811       ^821       ^831       ^841       ^851       ^861       ^871       ^881       ^891       

GCAGATATTC GTAATTATGC GGGCAACGTC TGGTATCAGC GCGAAGTCTT TATACCGAAA GGTTGGGCAG GCCAGCGTAT CGTGCTGCGT TTCGATGCGG 
^901       ^911       ^921       ^931       ^941       ^951       ^961       ^971       ^981       ^991       

TCACTCATTA CGGCAAAGTG TGGGTCAATA ATCAGGAAGT GATGGAGCAT CAGGGCGGCT ATACGCCATT TGAAGCCGAT GTCACGCCGT ATGTTATTGC 
^1001      ^1011      ^1021      ^1031      ^1041      ^1051      ^1061      ^1071      ^1081      ^1091      

CGGGAAAAGT GTACGTATCA CCGTTTGTGT GAACAACGAA CTGAACTGGC AGACTATCCC GCCGGGAATG GTGATTACCG ACGAAAACGG CAAGAAAAAG 
^1101      ^1111      ^1121      ^1131      ^1141      ^1151      ^1161      ^1171      ^1181      ^1191      

CAGTCTTACT TCCATGATTT CTTTAACTAT GCCGGAATCC ATCGCAGCGT AATGCTCTAC ACCACGCCGA ACACCTGGGT GGACGATATC ACCGTGGTGA 
^1201      ^1211      ^1221      ^1231      ^1241      ^1251      ^1261      ^1271      ^1281      ^1291      

CGCATGTCGC GCAAGACTGT AACCACGCGT CTGTTGACTG GCAGGTGGTG GCCAATGGTG ATGTCAGCGT TGAACTGCGT GATGCGGATC AACAGGTGGT 
^1301      ^1311      ^1321      ^1331      ^1341      ^1351      ^1361      ^1371      ^1381      ^1391      

TGCAACTGGA CAAGGCACTA GCGGGACTTT GCAAGTGGTG AATCCGCACC TCTGGCAACC GGGTGAAGGT TATCTCTATG AACTGTGCGT CACAGCCAAA 
^1401      ^1411      ^1421      ^1431      ^1441      ^1451      ^1461      ^1471      ^1481      ^1491      

AGCCAGACAG AGTGTGATAT CTACCCGCTT CGCGTCGGCA TCCGGTCAGT GGCAGTGAAG GGCGAACAGT TCCTGATTAA CCACAAACCG TTCTACTTTA 
^1501      ^1511      ^1521      ^1531      ^1541      ^1551      ^1561      ^1571      ^1581      ^1591      

CTGGCTTTGG TCGTCATGAA GATGCGGACT TACGTGGCAA AGGATTCGAT AACGTGCTGA TGGTGCACGA CCACGCATTA ATGGACTGGA TTGGGGCCAA 
^1601      ^1611      ^1621      ^1631      ^1641      ^1651      ^1661      ^1671      ^1681      ^1691      

CTCCTACCGT ACCTCGCATT ACCCTTACGC TGAAGAGATG CTCGACTGGG CAGATGAACA TGGCATCGTG GTGATTGATG AAACTGCTGC TGTCGGCTTT 
^1701      ^1711      ^1721      ^1731      ^1741      ^1751      ^1761      ^1771      ^1781      ^1791      

AACCTCTCTT TAGGCATTGG TTTCGAAGCG GGCAACAAGC CGAAAGAACT GTACAGCGAA GAGGCAGTCA ACGGGGAAAC TCAGCAAGCG CACTTACAGG 
^1801      ^1811      ^1821      ^1831      ^1841      ^1851      ^1861      ^1871      ^1881      ^1891      

CGATTAAAGA GCTGATAGCG CGTGACAAAA ACCACCCAAG CGTGGTGATG TGGAGTATTG CCAACGAACC GGATACCCGT CCGCAAGGTG CACGGGAATA 
^1901      ^1911      ^1921      ^1931      ^1941      ^1951      ^1961      ^1971      ^1981      ^1991      

TTTCGCGCCA CTGGCGGAAG CAACGCGTAA ACTCGACCCG ACGCGTCCGA TCACCTGCGT CAATGTAATG TTCTGCGACG CTCACACCGA TACCATCAGC 
^2001      ^2011      ^2021      ^2031      ^2041      ^2051      ^2061      ^2071      ^2081      ^2091      

GATCTCTTTG ATGTGCTGTG CCTGAACCGT TATTACGGAT GGTATGTCCA AAGCGGCGAT TTGGAAACGG CAGAGAAGGT ACTGGAAAAA GAACTTCTGG 
^2101      ^2111      ^2121      ^2131      ^2141      ^2151      ^2161      ^2171      ^2181      ^2191      

CCTGGCAGGA GAAACTGCAT CAGCCGATTA TCATCACCGA ATACGGCGTG GATACGTTAG CCGGGCTGCA CTCAATGTAC ACCGACATGT GGAGTGAAGA 
^2201      ^2211      ^2221      ^2231      ^2241      ^2251      ^2261      ^2271      ^2281      ^2291      

GTATCAGTGT GCATGGCTGG ATATGTATCA CCGCGTCTTT GATCGCGTCA GCGCCGTCGT CGGTGAACAG GTATGGAATT TCGCCGATTT TGCGACCTCG 
^2301      ^2311      ^2321      ^2331      ^2341      ^2351      ^2361      ^2371      ^2381      ^2391      

CAAGGCATAT TGCGCGTTGG CGGTAACAAG AAAGGGATCT TCACTCGCGA CCGCAAACCG AAGTCGGCGG CTTTTCTGCT GCAAAAACGC TGGACTGGCA 
^2401      ^2411      ^2421      ^2431      ^2441      ^2451      ^2461      ^2471      ^2481      ^2491      

TGAACTTCGG TGAAAAACCG CAGCAGGGAG GCAAACAATG A
^2501      ^2511      ^2521      ^2531      ^2541

Warning: available plasmid sequence is exactly same as the mRNA sequence. It seems we never managed to reconstruct the original plasmid sequence used to express the studied mRNA.

Credibility of mRNA sequence:

reverse_engineered_fragment_and_the_rest_is_a_guess

The name of the plasmid:
H-CP-IREScp-GUS The name of the promoter used to express this mRNA:
T7

Aliases of the plasmid name:

Alias: hCP-IREScp-GUS

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

not tested

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

not tested

A promoter reported in cDNA corresponding to IRES sequence:

not tested

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

The origin of IRES in the plasmid:

viral

The donor organism of the IRES segment:

Crucifer tobamovirus

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

gggcgaattg ggtaccgggc cccccctcga ggtcgacggt atcgataccg tcgacctcga gggggggccc ggtaccATGT CTTACAACAT TACAAACCCG 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

AATCAGTACC AGTATTTCGC AGCGGTGTGG GCAGAGCCCA TTCCGATGCT TAACCAGTGC ATATCGGCAT TGTCACAATC GTACCAAACA CAAGCTGCGA 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

GAGACACTGT TAGACAGCAA TTCTCAAACT TGTTGAGTGC GGTTGTGGCT CCGAGCCAGC GGTTCCCAGA AACAGGGTCC CGGGTGTATG TTAACTCGGC 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

TGTTATAAAG CCGTTGTACG AGGCTCTTAT GAAGTCCTTT GATACTAGAA ATAGGATCAT AGAGACGGAA GAGGAGTCAC GCCCGTCAGC TTCCGAAGTA 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

CGTAACGCGA CACAACGTGT TGATGATGCG ACCGTTTCCA TTAGAAGTCA AATTCAGCTG TTGCTGAGCG AGCTTTCCAG TGGACATGGT TATATGAACA 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

GGGCAGAGTT CGAGGCTTTG GTGCCATGGA CTACTGCTGC TGCTACTTAG aagcttatcg atttcgaacc cggggtaccg aattcgtcga ttcggttgca 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

gcatttaaag cggttgacaa ctttaaaaga aggaaaaaga aggttgaaga aaagggtgta gtaagtaagt ataagtacag accggagaag tacgccggtc 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

ctgattcgtt taatttgaaa gaagaaaccA TGGTCCGTCC TGTAGAAACC CCAACCCGTG AAATCAAAAA ACTCGACGGC CTGTGGGCAT TCAGTCTGGA 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

TCGCGAAAAC TGTGGAATTG ATCAGCGTTG GTGGGAAAGC GCGTTACAAG AAAGCCGGGC AATTGCTGTG CCAGGCAGTT TTAACGATCA GTTCGCCGAT 
^801       ^811       ^821       ^831       ^841       ^851       ^861       ^871       ^881       ^891       

GCAGATATTC GTAATTATGC GGGCAACGTC TGGTATCAGC GCGAAGTCTT TATACCGAAA GGTTGGGCAG GCCAGCGTAT CGTGCTGCGT TTCGATGCGG 
^901       ^911       ^921       ^931       ^941       ^951       ^961       ^971       ^981       ^991       

TCACTCATTA CGGCAAAGTG TGGGTCAATA ATCAGGAAGT GATGGAGCAT CAGGGCGGCT ATACGCCATT TGAAGCCGAT GTCACGCCGT ATGTTATTGC 
^1001      ^1011      ^1021      ^1031      ^1041      ^1051      ^1061      ^1071      ^1081      ^1091      

CGGGAAAAGT GTACGTATCA CCGTTTGTGT GAACAACGAA CTGAACTGGC AGACTATCCC GCCGGGAATG GTGATTACCG ACGAAAACGG CAAGAAAAAG 
^1101      ^1111      ^1121      ^1131      ^1141      ^1151      ^1161      ^1171      ^1181      ^1191      

CAGTCTTACT TCCATGATTT CTTTAACTAT GCCGGAATCC ATCGCAGCGT AATGCTCTAC ACCACGCCGA ACACCTGGGT GGACGATATC ACCGTGGTGA 
^1201      ^1211      ^1221      ^1231      ^1241      ^1251      ^1261      ^1271      ^1281      ^1291      

CGCATGTCGC GCAAGACTGT AACCACGCGT CTGTTGACTG GCAGGTGGTG GCCAATGGTG ATGTCAGCGT TGAACTGCGT GATGCGGATC AACAGGTGGT 
^1301      ^1311      ^1321      ^1331      ^1341      ^1351      ^1361      ^1371      ^1381      ^1391      

TGCAACTGGA CAAGGCACTA GCGGGACTTT GCAAGTGGTG AATCCGCACC TCTGGCAACC GGGTGAAGGT TATCTCTATG AACTGTGCGT CACAGCCAAA 
^1401      ^1411      ^1421      ^1431      ^1441      ^1451      ^1461      ^1471      ^1481      ^1491      

AGCCAGACAG AGTGTGATAT CTACCCGCTT CGCGTCGGCA TCCGGTCAGT GGCAGTGAAG GGCGAACAGT TCCTGATTAA CCACAAACCG TTCTACTTTA 
^1501      ^1511      ^1521      ^1531      ^1541      ^1551      ^1561      ^1571      ^1581      ^1591      

CTGGCTTTGG TCGTCATGAA GATGCGGACT TACGTGGCAA AGGATTCGAT AACGTGCTGA TGGTGCACGA CCACGCATTA ATGGACTGGA TTGGGGCCAA 
^1601      ^1611      ^1621      ^1631      ^1641      ^1651      ^1661      ^1671      ^1681      ^1691      

CTCCTACCGT ACCTCGCATT ACCCTTACGC TGAAGAGATG CTCGACTGGG CAGATGAACA TGGCATCGTG GTGATTGATG AAACTGCTGC TGTCGGCTTT 
^1701      ^1711      ^1721      ^1731      ^1741      ^1751      ^1761      ^1771      ^1781      ^1791      

AACCTCTCTT TAGGCATTGG TTTCGAAGCG GGCAACAAGC CGAAAGAACT GTACAGCGAA GAGGCAGTCA ACGGGGAAAC TCAGCAAGCG CACTTACAGG 
^1801      ^1811      ^1821      ^1831      ^1841      ^1851      ^1861      ^1871      ^1881      ^1891      

CGATTAAAGA GCTGATAGCG CGTGACAAAA ACCACCCAAG CGTGGTGATG TGGAGTATTG CCAACGAACC GGATACCCGT CCGCAAGGTG CACGGGAATA 
^1901      ^1911      ^1921      ^1931      ^1941      ^1951      ^1961      ^1971      ^1981      ^1991      

TTTCGCGCCA CTGGCGGAAG CAACGCGTAA ACTCGACCCG ACGCGTCCGA TCACCTGCGT CAATGTAATG TTCTGCGACG CTCACACCGA TACCATCAGC 
^2001      ^2011      ^2021      ^2031      ^2041      ^2051      ^2061      ^2071      ^2081      ^2091      

GATCTCTTTG ATGTGCTGTG CCTGAACCGT TATTACGGAT GGTATGTCCA AAGCGGCGAT TTGGAAACGG CAGAGAAGGT ACTGGAAAAA GAACTTCTGG 
^2101      ^2111      ^2121      ^2131      ^2141      ^2151      ^2161      ^2171      ^2181      ^2191      

CCTGGCAGGA GAAACTGCAT CAGCCGATTA TCATCACCGA ATACGGCGTG GATACGTTAG CCGGGCTGCA CTCAATGTAC ACCGACATGT GGAGTGAAGA 
^2201      ^2211      ^2221      ^2231      ^2241      ^2251      ^2261      ^2271      ^2281      ^2291      

GTATCAGTGT GCATGGCTGG ATATGTATCA CCGCGTCTTT GATCGCGTCA GCGCCGTCGT CGGTGAACAG GTATGGAATT TCGCCGATTT TGCGACCTCG 
^2301      ^2311      ^2321      ^2331      ^2341      ^2351      ^2361      ^2371      ^2381      ^2391      

CAAGGCATAT TGCGCGTTGG CGGTAACAAG AAAGGGATCT TCACTCGCGA CCGCAAACCG AAGTCGGCGG CTTTTCTGCT GCAAAAACGC TGGACTGGCA 
^2401      ^2411      ^2421      ^2431      ^2441      ^2451      ^2461      ^2471      ^2481      ^2491      

TGAACTTCGG TGAAAAACCG CAGCAGGGAG GCAAACAATG A
^2501      ^2511      ^2521      ^2531      ^2541

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:
CP

The description of the protein encoded in this ORF:
coat 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):

77-550

ORF

ORF position:

2

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

The abbreviated name of this ORF/gene:
GUS

The description of the protein encoded in this ORF:
beta-glucuronidase

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):

730-2541

Remarks:

A single-read sequencing result between CP and GUS ORFs obtained from T. Komarova.

Citations:

Ivanov P. A., Karpova O. V., Skulachev M. V., Tomashevskaya O. L., Rodionova N. P., Dorokhov YuL, Atabekov J. G. (1997) A tobamovirus genome that contains an internal ribosome entry site functional in vitro. Virology. 232(1):32-43

IRESs:

IRES:

Version: `2`	Last change: `2009-08-26 13:44:05`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš
The IRES name: `crTMV_IREScp`	The functional status of IRES: `functional`

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

580-727

How IRES boundaries were determined:
experimentally_determined

5'-end of IRES relative to last base of the STOP codon of the upstream ORF: `30`	3'-end of IRES relative to last base of the STOP codon of the upstream ORF: `177`
5'-end of IRES relative to first base of the START codon of the downstream ORF: `-150`	3'-end of IRES relative to first base of the START codon of the downstream ORF: `-3`

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

gaattcgtcg attcggttgc agcatttaaa gcggttgaca actttaaaag aaggaaaaag aaggttgaag aaaagggtgt agtaagtaag tataagtaca 
^580       ^590       ^600       ^610       ^620       ^630       ^640       ^650       ^660       ^670       

gaccggagaa gtacgccggt cctgattcgt ttaatttgaa agaagaaa
^680       ^690       ^700       ^710       ^720

Citations:

The translation experiments:

Translation results:

IRESite Translation Id: 632

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

The translation method used to study IRES function:
in vitro The in vitro translation system:
wheat germ lysate

The organism used for translation:

The temperature (in degrees of Celsia):
30

The relative translation efficiency in % of this IRES:

100.000

	Name of the plasmid used as the negative control. `H-CP-TMV_UI-GUS`
	`IRESite Id` of the plasmid used as negative control. `604`
	The relative translation efficiency in % of the negative control: `0`
	The size (length) of intercistronic region in the negative control: `80`

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

Rapamycin affects translation:
not tested

Type of RNA subject to translation:

exogenous_RNA_without_cap_without_polyA_tail

Remarks:

Data reflect results shown in Figure 7B. However, IRESite did not quantify intensities of the bands.

Citations:

Translation results:

IRESite Translation Id: 633

Version: `1`	Last change: `2009-08-23 20:26:04`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš

The translation method used to study IRES function:
in vitro The in vitro translation system:
rabbit reticulocytes lysate

The organism used for translation:

The temperature (in degrees of Celsia):
37

The relative translation efficiency in % of this IRES:

100.000

	Name of the plasmid used as the negative control. `H-CP-TMV_UI-GUS`
	`IRESite Id` of the plasmid used as negative control. `604`
	The relative translation efficiency in % of the negative control: `0`
	The size (length) of intercistronic region in the negative control: `80`

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

Rapamycin affects translation:
not tested

Type of RNA subject to translation:

exogenous_RNA_without_cap_without_polyA_tail

Remarks:

Data reflect results shown in Figure 7B. However, IRESite did not quantify intensities of the bands.

Only GUS protein is produced from this message. Removal of the hairpin from the 5'-end (CP-IREScp-GUS plasmid)
results in synthesis of the CP protein while almost disabling the IRES-mediated synthesis of GUS.

Citations:

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