Promoters for gene expression

The present invention provides a novel expression system for the production of protein in bacterial hosts. The system utilizes novel promoters that are highly efficient in initializing transcription and therefore enhance protein yield. The promoters comprise the -35 region of the consensus E. coli promoter, the -10 region of the lppP-5 promoter and a spacer between these two regions derived from either the lpp or lacUV5 promoters.

FIELD OF THE INVENTION 
This invention relates to the art of genetic engineering as applied to 
bacterial hosts. More particularly, to expression systems for production 
of proteins in bacterial hosts. 
BACKGROUND OF THE INVENTION 
The advent of recombinant DNA technology has enabled the production of 
various naturally occurring and synthetic proteins in organisms such as 
bacteria, fungi, yeast and mammalian cells. In general it involves the 
insertion of genes that encode a desired protein into a host organism, and 
utilizing the host's cellular machinery to express the gene. 
Recombinant DNA technology is continually developing to achieve production 
of proteins in commercially acceptable yields. A limiting factor in 
recombinant production of proteins is the rate at which the gene encoding 
the desired protein is expressed. In particular, it has been found that 
the promoter region of a gene is critical in the transcription process of 
gene expression. An efficient promoter such as the trp promoter found in 
E. coli, binds tightly to DNA-directed RNA polymerase to initiate 
transcription of the gene in generating mRNA. A less efficient promoter 
such as the lac promoter binds RNA polymerase less tightly, resulting in a 
lower rate of mRNA generation. 
The trp promoter has been widely used in the production of heterologous 
proteins because of its ability to initiate transcription. Despite its 
efficiency, an inherent shortcoming of the trp promoter is that it is not 
easily controlled. Specifically, the trp promoter is not fully 
repressible, i.e. it can drive transcription before the host is grown in 
culture to a phase appropriate for protein production. Another widely used 
promoter is lac which is less efficient than trp, however is more 
controllable. 
To develop more efficient promoters, functional components of different 
promoters have been combined, for instance those described in U.S. Pat. 
No. 5,362,646. In one example, portions of the phage T7 promoter A.sub.1 
(P.sub.A1) were combined with two lac operators. Specifically, the spacer 
region between the so called -35 and -10 regions of the T7 promoter was 
replaced with a modified lac operator sequence, and to control the 
resulting promoter hybrid, a second lac operator was introduced 
downstream. The resulting promoter/operator system which is incorporated 
on the commercially available pUHE plasmids was found to initiate 
transcription efficiently upon induction and yet is highly repressed 
before induction. 
Another promoter described by Tsung et al (Proc. Natl. Acad. Sci. USA, 
1990, 87:5940) comprises the efficient trp -35 region, the -10 region from 
the highly efficient lppP-5 promoter (a variant of lpp promoter) and a 
spacer derived from the lac promoter. This promoter was shown to be so 
highly efficient in initiating transcription as to result in cell 
lethality. 
While various promoters have allowed improved yields of proteins in 
microbial hosts, there still remains a need for promoters that drive 
production of commercially valued proteins more efficiently. 
SUMMARY OF THE INVENTION 
According to an aspect of the present invention, there is provided a novel 
recombinant DNA construct useful for expressing a protein in a bacterial 
host. The construct comprises a coding region for a protein and linked 
operably therewith, a control region comprising a promoter having a DNA 
sequence selected from: 
##STR1## 
In another aspect, there is provided a novel expression vector that 
incorporates the constructs of the invention for use in obtaining 
bacterial host cell transformants that efficiently express the DNA coding 
for a protein of interest. 
##STR2##

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides DNA sequences useful in driving DNA 
expression with high efficiency in bacterial hosts such as E. coli. The 
use of expression vectors comprising these sequences provides a valuable 
means for achieving increased production of expressible proteins, both 
endogenous and heterologous. In one aspect of the invention, there is 
provided a novel recombinant DNA construct useful for expressing a protein 
in a bacterial host. The construct comprises a coding region for the 
protein operably linked with a control region comprising a promoter to 
enable expression of said protein in the host, wherein the promoter 
comprises a DNA sequence selected from: 
##STR3## 
These promoters have in common a -35 region of the consensus sequence 
TTGACA and a -10 region of the sequence TATACT. The spacer sequence, i.e. 
the sequence of 18 bases which is intervening, can according to the 
invention be either the sequence ACATAAAAAACTTTGTGT [SEQ ID NO:3] or 
CTTTATGCTTCCGGCTCG [SEQ ID NO:4] and more preferably the sequence 
ACATAAAAAACTTTGTGT[SEQ ID NO:3]. Accordingly, the invention provides in a 
preferred embodiment, DNA constructs in which DNA coding for a desired 
protein is linked operably under expression control to a promoter of the 
sequence 5'-TTGACAACATAAAAAACTTTGTGTTATACT-3' [SEQ ID NO:1]. 
Those skilled in the art will appreciate that the promoters of the 
invention constitute an essential one of the components required within 
the region functional to drive expression. and can be inserted using 
standard procedures into any suitable expression vector which can 
replicate in Gram -ve or +ve bacteria. More particularly, and to form a 
gene expression control region, the present promoters will be incorporated 
with such other control elements as are typically required for that 
expression, including a ribosome binding site and in embodiments of the 
invention, an operator that functions to control promoter function. These 
components are necessarily arranged relative to each other as required for 
expression to occur according to well understood principles of gene 
expression. 
In an embodiment of the invention, the control region of the construct 
incorporates an operator. Operators that can used include all that are 
directly inducible by chemical inducers. Examples of operators which are 
directly inducible include lactose, galactose, tryptophan and tetracycline 
operators (see Miller et al "The Operon" , Cold Spring Harbour Laboratory, 
1980 and Hillen et al, J. Mol. Biol., 1984, 172:185). Preferred operators 
are highly repressible so that expression of DNA coding for the protein 
can be controlled. In a specific embodiment, the control region comprises 
the lac operator (FIG. 1) which prevents expression from the promoter in 
the absence of inducer isopropyl-.beta.-D-thiogalactopyranoside (IPTG). 
The control region further comprises a ribosome binding site (RBS) sequence 
to facilitate the binding of ribosomes to the mRNA transcript and thereby 
initiate the translation of the RNA coding region to generate the protein. 
Suitable ribosome binding sites include lac, T5, In a preferred embodiment 
the RBS is a sequence derived from the T5 phage RBS having the sequence 
5'-ATTAAAGAGGAGAAATTAAGC-3' [SEQ ID NO:5]. 
The control region of constructs according to the present invention are 
operably linked with a coding region for an endogenous or heterologous 
proteins. By the term "heterologous protein" is meant a polypeptide or 
protein which, although not naturally produced by the bacterial host, is 
expressed by this host when suitably transformed with DNA coding for the 
protein, such as genomic DNA, cDNA and synthetic DNA. Among the proteins 
that may be produced using the system herein described include, but are 
not limited to, hormones such as parathyroid hormone (PTH), glucagon or 
fragments thereof such as GLP-1 and GLP-2; growth factors such as 
epidermal growth factor (EGF); and lymphokines such as interleukin-6 and 
-8 (IL-6, -8). In order for isolation of the authentic form of the 
protein, i.e. protein without an additional N-terminal Met residue, fusion 
proteins may also be produced which are cleaved subsequent to expression. 
For example, DNA encoding a protein may be preceded by DNA encoding a 
signal peptide, such as the E. coli outer membrane protein ompA. In this 
instance the expressed gene yields a fusion protein comprising a Met 
residue followed by the ompA signal peptide which is followed by the 
desired protein. The signal peptide carries the fusion protein through the 
intermembrane of the bacterium where the signal peptide is cleaved. Other 
signal peptides which may be used include alkaline phosphatase and protein 
A from Streptococcus. Alternatively, a fusion protein may be synthesized 
and cleaved in a separate procedure to yield the desired protein. For 
example glutathione-S-transferase (GST) may be cleaved from a desired 
protein with thrombin or factor Xa. 
In a specific embodiment of the invention, the coding region comprises DNA 
encoding human PTH, the amino acid sequence of which is described by Hendy 
et al (Proc. Natl. Acad. Sci. USA, 1981, 78:7365). In the examples herein 
described, the DNA sequence coding for PTH was immediately preceded in 
reading frame with the ompA signal peptide. 
The preferred recombinant DNA constructs illustrated in FIG. 1, having the 
lpp or lacUV5 spacer, were produced from a single-stranded oligonucleotide 
synthesized by the phosphoramidite method. The gel-purified strand 
comprising the sequences from the XhoI to EcoRI restriction site was then 
used as a initial PCR target and was PCR amplified into a double stranded 
DNA fragment using complementary single stranded DNA oligonucleotides 
which hybridized specifically to the ends of either the initial 
oligonucleotide sequence shown or its complementary strand. Thus, the 
constructs are prepared using standard gene synthesis methodology, as 
described for example by Maniatus ("Molecular Cloning" Cold Spring Harbour 
Laboratories, 1982) and Innis et al ("PCR Protocols, A Guide to Methods 
and Applications"). 
In another aspect of the invention there is provided expression vectors 
useful for producing bacterial host cell transformants which incorporate a 
recombinant DNA construct according to the invention. DNA constructs 
according to the invention may be incorporated as a "cassette" into a 
vector, preferably a plasmid vector, by established techniques. Generally, 
a vector is cleaved at restriction sites that correspond with restriction 
sites on either end on the cassette. The cassette is then introduced by 
ligating the ends to the complementary cleaved sites on the vector. 
Although phage vectors can be use, plasmid vectors are preferred such as 
the pUC series of plasmids. Once incorporated on a suitable vector, the 
resulting plasmid may be amplified in a host to provide amounts sufficient 
for subsequent cloning work. It will be appreciated that DNA coding for 
the selected protein is conveniently incorporated on the plasmid with 
multiple cloning sites provided thereon, using standard cloning/ligation 
methods. Also, a plasmid will necessarily incorporate an origin of 
replication and most desirably will incorporate a marker such as the 
ampicillin or tetracycline resistance genes to allow the selection of 
transformed cells. 
Once DNA coding for the desired protein is incorporated on the vector, a 
selected bacterial host is transformed therewith using standard calcium 
chloride mediated transformation techniques. Suitable bacterial hosts 
include gram negative bacteria such E.coli and Salmonella. Preferably the 
host is a commercially available E. coli strain and most preferably JM101 
and derivatives thereof. 
When the controlling region of the DNA construct comprises the lac 
operator, as described in more detail hereinafter, the transformed host 
strain should be capable of expressing, preferably over-producing, the 
lacI product so that promoter function and hence expression of the 
protein, can be regulated. The need for lacI overproduction by the 
transformant can be met, according to one embodiment of the invention, by 
using hosts that already harbour the lacI.sup.q gene responsible for 
overproduction of the lacI product. LacI over-producing strains of E. coli 
that may be employed as host include the JM series of strains available 
from Clontech Laboratories Inc., Calif., USA. Specific host strains 
suitable for use include JM101, JM105 and JM107. 
The need for lacI overproduction in the transformant may alternatively be 
met by incorporating the lacI.sup.q gene on vectors of the invention. 
Since, in this situation, the overproduction of lacI is mediated by the 
vector, any of a variety of commercially available bacterial host strains 
may be employed, including E. coli strains DH1, RR1, C600, CMK603 and 
EB505. The lacI.sup.q gene to be incorporated on the vector may be 
obtained as a 1.2 kb HindIII fragment of plasmid pMMB22 (described by 
Bagdasarian et al (Gene, 1983, 26:273) and then incorporated 
non-disruptively at any site on the plasmid vector. 
To enhance the stability of inheritance of vectors, in particular plasmids, 
from the strain originally transformed to its progeny, a partition element 
(par) functional in E. coli may also be incorporated on the vector. One 
such par element may be liberated from pSC101 as a 380bp HincII/AvaI 
fragment and then cloned into a suitable site on the vector. 
Following transformation, bacterial hosts harbouring the excretion vector 
are cultured in a culturing medium most appropriate for the selected host. 
For E. coli, LB broth or 2YT medium (yeast extract/tryptone) can be used 
to culture those strains herein preferred. Selective pressure for plasmid 
transformants should be maintained by providing a cytotoxic agent which 
kills the untransformed host strain. 
For example, a transformant with a plasmid harbouring the gene for 
tetracycline resistance should be cultured in medium containing 
tetracycline. Medium concentrations of tetracycline around 5-15.mu.g/mL 
are suitable. 
The promoter on the construct is preferably regulatable through binding of 
a repressor molecule to an operator located adjacent to the promoter in 
the control region. In a preferred embodiment, the lacI gene product binds 
to a lac operator located adjacent the promoter. In this instance, binding 
of lacI product represses the promoter, lowering expression levels of 
coding DNA under its control. To raise expression levels, the chemical 
IPTG (isopropyl-.beta.-D-thiogalactopyranoside), which binds the lacI and 
derepresses the promoter, is added to the culture medium to derepress the 
promoter and induce expression. Suitably, IPTG is added to the culture 
medium when the cells have reached mid log growth phase. 
To determine the optimum density to which cultures should be grown to 
realize maximum yield of the desired protein, trials can be conducted and 
protein levels assayed in a time-course experiment. In general, reasonable 
yields of protein may be recovered once cells reach mid log phase, 
although greater amounts of protein can be expected to accumulate within 
about 4-5 hours thereafter. 
The desired protein can be purified by techniques established in the art as 
being appropriate for that protein. In a specific embodiment of the 
invention, expressed PTH is excreted beyond the periplasmic space and into 
the culture medium where it is recovered directly. When protein is 
excreted, the spent medium can be isolated using biochemical techniques 
that reflect the nature of the protein in terms of its molecular size, net 
charge, isoelectric point, et. The medium may be concentrated first such 
as by lyophilization. Further, when antibodies are available or a natural 
ligand for the protein is available, affinity columns may be use. 
Specific embodiments of the invention are now exemplified with reference to 
the drawings. 
EXAMPLE 1 
In its mature form, PTH is an 84-amino acid peptide that acts in humans to 
raise blood calcium and increase bone resorption. DNA coding for a PTH 
analogue, bearing an N-terminal methionine residue was synthesized using 
the established techniques and according to the amino acid sequence 
published by Hendy et al., supra. 
Preferred recombinant DNA constructs incorporating promoter #1 and #2 as 
well as reference promoters #3 and #4, illustrated in FIG. 1, to which 
reference is now made were produced from a single-stranded oligonucleotide 
synthesized by the phosphoramidite method. The gel-purified strand 
comprising the sequences from the XhoI to EcoRI restriction site was then 
used as a initial PCR target and was PCR amplified into a double stranded 
DNA fragment using complementary single stranded DNA oligonucleotides 
which hybridized specifically to the ends of either the initial 
oligonucleotide sequence shown or its complementary strand. Thus, the 
constructs are prepared using standard gene synthesis methodology, as 
described for example by Maniatus ("Molecular Cloning" Cold Spring Harbour 
Laboratories, 1982) and Innis et al ("PCR Protocols, A Guide to Methods 
and Applications"). 
The constructs were then cloned into a pUC18 derived plasmid which confers 
tetracycline resistance in place of ampicillin resistance. A JM101 derived 
E. coli host strain was then transfected according established techniques 
(see Maniatus et al "Molecular Cloning", Cold Spring Harbour Laboratory, 
1982) 
EXAMPLE 2 
Expression of Transformed Host 
The transformants containing the PTH vectors were cultured overnight at 
30.degree. C. in 2YT broth containing 0.5% glucose and tetracycline and 
then inoculated into fresh medium of the same composition, with continued 
culturing at 30.degree. C. until reaching mid log phase. Cultures were 
then induced (1mM IPTG) at 1 hour growth intervals, aliquots of culture 
were withdrawn and fractionated to produce samples of culture medium to 
identify excreted PTH products using a standard Allegro assay. The results 
of these assays are provided in Table 1 below: 
TABLE 1 
______________________________________ 
Promoter Max PTH (mg/L) 
# -35 region 
spacer -10 region 
RBS 6-8 hrs 
______________________________________ 
1 trp lpp lppP-5 T5 245 
lac 148 
2 trp lacUV5 lppP-5 T5 121 
lac 10 
3 trp lacO lppP-5 T5 50 
lac 5 
4 T7 lacO T7 T5 100 
lac 10 
______________________________________ 
Results of the Allegro assay indicate that the promoters incorporating the 
18 bp lpp and lacUV5 sequences facilitate enhanced levels of heterologous 
PTH protein. Promoter #1 and #2 compare favourably to promoter #3 wherein 
the spacer was substituted with a modified lac operator sequence (lacO); 
and promoter #4 wherein the -35 and -10 regions are from phage T7 and the 
spacer is iacO promoter. Studies with the same promoters expressing the 
gene coding for chloramphenicol acetyl transferase (CAT) showed similar 
results of enhanced expression for promoters #1 and #2. Also, it was noted 
that each of the promoters studied exhibited enhanced protein yield when 
combined with the T5 RBS in comparison to the lac derived RBS. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 13 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TTGACAACATAAAAAACTTTGTGTTATACT30 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
TTGACACTTTATGCTTCCGGCTCGTATACT30 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ACATAAAAAACTTTGTGT18 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CTTTATGCTTCCGGCTCG18 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ATTAAAGAGGAGAAATTAAGC21 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 136 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CTCGAGGCCACCCGGGCCAAAATTTATCAAAAATATCTGCAGTTGACAACATAAAAAACT60 
TTGTGTTATACTGTCGACAATTGTGAGCGGATAACAATTTCACACAGAATTCATTAAAGA120 
GGAGAAATTAAGCATG136 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 132 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CTCGAGGCCACCCGGGCCAAAATTTATCAAAAATATCTGCAGTTGACAACATAAAAAACT60 
TTGTGTTATACTGTCGACAATTGTGAGCGGATAACAATTTCACACAGAATTCAGGAGGAA120 
AAAATTATGATG132 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 136 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CTCGAGGCCACCCGGGCCAAAATTTATCAAAAATATCTGCAGTTGACACTTTATGCTTCC60 
GGCTCGTATACTGTCGACAATTGTGAGCGGATAACAATTTCACACAGAATTCATTAAAGA120 
GGAGAAATTAAGCATG136 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 132 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
CTCGAGGCCACCCGGGCCAAAATTTATCAAAAATATCTGCAGTTGACACTTTATGCTTCC60 
GGCTCGTATACTGTCGACAATTGTGAGCGGATAACAATTTCACACAGAATTCAGGAGGAA120 
AAAATTATGATG132 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 136 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
CTCGAGGCCACCCGGGCCAAAATTTATCAAAAATATCTGCAGTTGACATTGTGAGCGGAT60 
AACAATTATACTGTCGACAATTGTGAGCGGATAACAATTTCACACAGAATTCATTAAAGA120 
GGAGAAATTAAGCATG136 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 132 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CTCGAGGCCACCCGGGCCAAAATTTATCAAAAATATCTGCAGTTGACATTGTGAGCGGAT60 
AACAATTATACTGTCGACAATTGTGAGCGGATAACAATTTCACACAGAATTCAGGAGGAA120 
AAAATTATGATG132 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 101 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CTCGAGGCCACCCGGGCCAAAATTTATCAAATTGACAACATAAAAAACTTTGTGTTATAC60 
TGTCGACAATTGTGAGCGGATAACAATTTCACACAGAATTC101 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 101 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CTCGAGGCCACCCGGGCCAAAATTTATCAAATTGACACTTTATGCTTCCGGCTCGTATAC60 
TGTCGACAATTGTGAGCGGATAACAATTTCACACAGAATTC101 
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