BACKGROUND
along with the fulfilment of the whole genome sequencing of different organisms including many crops , the challenge of how to comprehensively study gene functions on a large scale emerges.
gene cloning, gene expression in different modes , protein localization and interaction, silencing the gene, and promoter function and regulation are always the issues a researcher concerns.
therefore, a series of high efficient cloning and expression vectors are developed in different labs  <cit> .
the primary cloning approaches available can be mainly classed into two groups: the traditional restriction enzyme/ligase  strategy including ta cloning technology and the dna recombinational cloning systems including the gateway cloning system from invitrogen and the creator cloning system from clontech.
the former restriction enzyme/ligase strategy, even though cheap, suffers from various problems, mainly no suitable restriction enzyme sites in binary vectors compatible for different genes and promoters.
therefore, it is rather limited for projects on a large scale and multiple functions.
ta cloning technology increases the cloning efficiency, and at the same time it increases the cost too, because it is difficult to generate a high-quality ta cloning vector in home-made and a researcher has to purchase different ta cloning kits from the market.
the dna recombinational cloning system, especially the gateway cloning system from invitrogen, is recently developed and widely used.
the first step of this system  is to clone the pcr product into a donor vector to produce an entry clone  <cit> .
once an entry clone is available, the gene of interest is easily transferred into different expression vectors through an lr reaction, which is a high efficient reaction.
due to its many advantages, many cloning systems related were developed  <cit> .
however, there are still some limitations for this technology.
for example, the primers at the first step need a long extra sequence for recombination reaction, which decreases the efficiency of pcr and bp reaction and increases the cost.
aimed at these problems, higher efficiency vector systems are developed.
invitrogen develops a high efficiency kit , which combines ta cloning and br reaction to eliminate the bp reaction, and the resulting vector can be directly used to lr reaction , but it is at the cost of a high price.
the zebata system introduces the ta cloning into plant binary vectors and results in zero background vectors  <cit> .
this zebata system obviously needs to make a lot of home-made and low efficiency t-binary-vectors for different purposes, such as to label a gene of interest with different tags.
ta cloning has another problem waiting to be solved: the gene orientation, which decreases the cloning efficiency.
multiple gateway systems are developed for cloning multiple dna fragments  into a expression vector in one step  <cit> , but it also suffers from low efficiency because of many recombination sites.
the type iis endonucleases recognize asymmetric dna sequences, 4– <dig> bp long, and cleave both strands at specific locations up to  <dig> bases away from their recognition site  <cit> .
recently, two excellent papers reported new gene cloning strategies, referred to golden gate cloning   <cit>  and goldenbraid   <cit> , with which it is possible to seamlessly assemble multiple reusable gene modules, including promoters, genes and terminators, together in a binary vector in a single restriction-ligation.
even though a lot of cloning systems are now available as discussed above, many common problems still exist in our routine experiment.
in modern functional genomic study, one of challenges is to construct a set of vectors for a gene of interest to study gene functions in special spatio-temporal patterns.
it includes cloning rapidly and efficiently, labeling proteins with different tags, shuffling randomly promoters and genes, introducing a desired regulatory element for a given promoter or gene, and modifying vectors in hand for ongoing demands.
therefore, the flexibility of vectors appears to be a main challenge.
what’s more, gateway technology available now leaves a common question to be answered: after lr reaction, there is a longer spacer existed between genes and tags, which may result in detrimental effect on protein functions  <cit> .
in this study, we developed a highly flexible expression system to cope with on-going demands in plant functional genomic study, based on classical cloning approaches other than to introduce a new method.
firstly, we employed widely-used site-specific recombination  cloning system  for high cloning efficiency.
secondly, we introduced two pairs of ssrs, one for genes and the other for promoters in two independent entry clones .
thirdly, we adopted traditional restriction enzyme/ligase  strategy in an easy-operating intermediate cloning vector, instead of a difficult-operating binary vector as previous researchers used.
this rel is designed for cloning of desired multiple regulatory elements or tags, and it also provide flexible choices for cloning.
RESULTS
through directly synthetizing sequences of all dna fragments  except the backbone of vectors indicated below, we developed our cloning system, biovector.
biovector is composed of three basic vectors: a gene entry clone  with one pair of ssrs of attl1/attl <dig>  a promoter entry clone  with another pair of ssrs of attl3/attl <dig>  and a binary destination vector  with two pairs of corresponding ssrs of attr1/attr <dig> and attr3/attr <dig>  therefore, with a single lr reaction , a promoter can be fused to a gene and a plant expression vector is constructed.
gene entry clone
the gene entry clone   was developed from the puc vector due to its advantage of easy-operating for cloning, and introduced up to  <dig> restriction enzyme/ligase  sites including t/a cloning sites , therefore it is convenient to clone any dna fragments and to assemble multiple dna fragments into a gec.
rel sites were inserted inside site-specific recombination  sites  in gecs, so that it was possible to make a seamless fusion between a gene and a tag in our system, rather than to introduce a ssr spacer  between them as widely-used gateway cloning system does, in which the tags are in destination vectors  <cit> .
such a spacer has the potential to result in the synthesis of a non-functional or insoluble protein-tag fusion  <cit> .
another improvement was that a stop code was put at the end of rel sites and inside ssr .
thus, only the gene without a stop code was needed to be cloned into a gec, then it can be removed from one gec to another by a simple rel.
in this case, there was no need to clone a gene for the n- or c-terminal fusion, respectively, as wide-used gateway cloning systems do  <cit> .
therefore, gecs saved both time and cost for cloning and sequencing of genes.
✶all vectors were delivered to abrc .
“n” or “c” indicates that the position of a tag is at the “n” or “c” terminus of a target gene, “n” or “c” for the “n” or “c” half part of yfp fluorescence protein.
all vectors contain recombination sites of attl <dig> and attl <dig> and have the chloramphenicol selection marker in e. coli.
all the sequences are showed in additional file  <dig>
therefore, genes, tags, and any regulatory elements of interest from pcr amplification can be directly cloned into gecs after digestion.
we constructed a set of alternative gecs for labeling a desired protein with different tags at the n- or c-terminus .
and all gecs shared identical rel sites, and it was easy to move a gene from one gec to another with a simple rel.
a ccdb gene in rel sites  served as a negative selection marker for e. coli dh5α as all gateway entry vectors employ   <cit> .
the chloramphenicol  was employed as a selection marker in e. coli, which may be compatible to most of binary vectors available.
we designed a primer entry-f  for forward sequencing, and the general primer sp <dig>  can be used as a reverse sequencing primer.
plenty of rel sites in gecs provide a flexible platform for ad arbitrium modifying the vector by researcher themselves for their own individual study.
this is an important respect of this flexible system, because the plasticity of a system is always demanded in functional genome study, but a shortage of wide-used gateway systems  <cit> .
for example, to insert rna-binding loops   <cit>  inside ssrs in gec makes it possible to label an rna.
one of our considerations was to develop gecs compatible with the present gateway destination vectors for plants, e. coli, and yeast, so researchers can keep their vectors in hands when introduce biovector.
promoter entry clone
most of gateway based cloning systems normally put a constitutive promoter, such as camv 35s, in the binary vector, in which the promoter is not easy to be replaced by another promoter due to its low cloning efficient.
even though multiple gateway system  <cit>  clone a promoter inside the specific ssr sites, it bears low efficient in subsequent multiple recombination reactions and it is also difficult to insert a cis-element next to the promoter.
the promoter entry clone  in biovector  also originated from puc vector, shared the same backbone as gec, and embraced up to  <dig> rel sites inside ssr sites .
the plenty of rel sites benefited to freely clone or assemble various promoters or cis-elements together to study their functions.
obviously, as in gecs, pecs were good to be updated by researchers for an ambitious idea.
there were three pecs available with no enhancer, one enhancer, or two enhancers, respectively .
thus, a gene can be expressed or over-expressed from a native promoter, and the bona fide functions of a gene could be revealed.
such a strategy was designed to avoid disadvantages of generally over-expressing promoter camv 35s, which has overt ectopic effects  <cit> , weak or null functions in some tissues and plants  <cit> , and adverse effects on adjacent genes  <cit> .
✶ all vectors were delivered to abrc .
all vectors contain recombination sites of attl <dig> and attl <dig> and have the chloramphenicol selection marker in e. coli.
all the sequences are showed in additional file  <dig>
again, the three pecs had identical rel sites, which conferred to easily shift promoters among them.
the selection marker and the sequencing primers in pecs were same to that of gecs.
besides rel strategy, other cloning technology, such as in-fusion® hd cloning from clontech , can be an alternative efficient approach for cloning genes or promoters into gecs or pecs, so as to increase the cloning efficiency.
if introducing the type iis endonucleases, such as bsa i, bbs i, and bsmb i, gecs and pecs could be compatible to golden gate cloning  <cit>  and goldenbraid  <cit> .
binary destination vector
all binary destination vectors  in this study were developed from pgreen/pclean, which are highly efficient in a wide arrange of plants  <cit> .
so, bdvs have smaller size  than most of gateway-compatible binary vectors   <cit> .
and the small size of binary vectors facilitates to increase the cloning efficiency, plasmid yield and plant transformation  <cit> .
because the right border of the t-dna is largely preserved whereas the left border is frequently truncated after integration  <cit> , a selectable marker in planta was placed next to the left border in bdvs to ensure that all transformants with a positive marker always carry the introduced gene.
a set of binary vectors were designed for a genomic gene  or cds gene expression , gene silencing , and ethanol-inducible expression , respectively.
there were more bdvs available with various selection markers in e. coli and plants , providing multiple choices in different projects.
so, biovector can be used to express a genomic gene spanning the sequence from the promoter to the terminator, to analyze the function of coding sequence from a desired promoter or ethanol-inducible promoter, to monitor proteins with fluorescent and other tags, to study protein-protein interaction, and to silence a gene in specific spatio-temporal mode.
the mcs in bdvs facilitates to be modified for extensive demands , such as replacing the preloaded transcription terminator or the selection marker.
e. coli
✶all vectors were delivered to abrc .
all the sequences are showed in additional file  <dig>
once a gene and a promoter were cloned into gecs or pecs, respectively, the gene and the promoter can be assembled into a binary vector through a single lr reaction , which is a high efficient and directional reaction .
several systems, such as multisite gateway cloning  <cit> , provide a possibility to combine promoters and genes at will, but with limited numbers of promoters and tags available.
biovector, however, made it easy to express a gene from different desired promoters with different intensities.
gene entry clones from wide-used gateway system made by most researchers shared the same ssr sites attl1/ <dig> at both ends with gecs.
thus, gene entry clones at hand were compatible with our bdvs, rather than discarding them when introduce biovector.
obviously, gecs and pces can serve as an intermediate vector for cloning due to plenty of mcss available .
in addition, gecs, pecs and bdvs can be shared worldwide as a library.
we have delivered all vectors in this study to abrc centre .
verification of biovector
with biovector we succeeded in stable or transient expression of gfp with or without nuclear localization signal  or nuclear export signal  in arabidopsis protoplasts , a gus gene from the companion cell-specific sucrose h+symporter <dig>  promoter  <cit>  with or without enhancers in arabidopsis , a myc-tagged gene in nicotiana benthamiana , and a luciferase gene under ethanol-inducible pattern n. benthamiana .
the results supported that biovector was efficient expression vector for plants.
CONCLUSIONS
perfect combination of conventional rel strategy and modern ssr technology confers obvious advantages to biovector.
exchangeable and efficient.
a gene and a promoter can be easily assembled together to fulfill expressing a gene from a temporal-spatio promoter with different intensity, especially overexpressing genes under the control of native promoters;  flexible.
gecs, pecs, and bdvs can be ad arbitrium modified with ongoing demands;  practical and versatile.
biovector can be applied to almost all fields in functional genome research of various plants;  universal and time-/labour-saving.
gecs can be efficiently applied to any plant, yeast, and e. coli destination vectors sharing corresponding ssr sites, and it is possible to construct a worldwide library as shared community resource for gecs, pecs and bdvs;  seamless fusion.
it is possible to make seamless fusion between a protein and a tag, rather than to introduce a detrimental ssr spacer as the widely-used gateway recombination system does;  broad application and interest.
the idea of biovector can also be applied to similar study in animals and yeast.

 along with the fulfilment of the whole genome sequencing of different organisms including many crops, the challenge of how to comprehensively study gene functions on a large scale emerges. gene cloning, gene expression in different modes, protein localization and interaction, silencing the gene, and promoter function and regulation are always the issues a researcher concerns. therefore, a series of high efficient cloning and expression vectors are developed in different labs. the primary cloning approaches available can be mainly classed into two groups: the traditional restriction enzyme/ligase strategy including ta cloning technology and the dna recombinational cloning systems including the gateway cloning system from invitrogen and the creator cloning system from clontech. the former restriction enzyme/ligase strategy, even though cheap, suffers from various problems, mainly no suitable restriction enzyme sites in binary vectors compatible for different genes and promoters. therefore, it is rather limited for projects on a large scale and multiple functions. ta cloning technology increases the cloning efficiency, and at the same time it increases the cost too, because it is difficult to generate a high-quality ta cloning vector in home-made and a researcher has to purchase different ta cloning kits from the market. the dna recombinational cloning system, especially the gateway cloning system from invitrogen, is recently developed and widely used. the first step of this system is to clone the pcr product into a donor vector to produce an entry clone. once an entry clone is available, the gene of interest is easily transferred into different expression vectors through an lr reaction, which is a high efficient reaction. due to its many advantages, many cloning systems related were developed. however, there are still some limitations for this technology. for example, the primers at the first step need a long extra sequence for recombination reaction, which decreases the efficiency of pcr and bp reaction and increases the cost. aimed at these problems, higher efficiency vector systems are developed. invitrogen develops a high efficiency kit, which combines ta cloning and br reaction to eliminate the bp reaction, and the resulting vector can be directly used to lr reaction, but it is at the cost of a high price. the zebata system introduces the ta cloning into plant binary vectors and results in zero background vectors. this zebata system obviously needs to make a lot of home-made and low efficiency t-binary-vectors for different purposes, such as to label a gene of interest with different tags. ta cloning has another problem waiting to be solved: the gene orientation, which decreases the cloning efficiency. multiple gateway systems are developed for cloning multiple dna fragments into a expression vector in one step, but it also suffers from low efficiency because of many recombination sites. the type iis endonucleases recognize asymmetric dna sequences, 4– bp long, and cleave both strands at specific locations up to bases away from their recognition site. recently, two excellent papers reported new gene cloning strategies, referred to golden gate cloning and goldenbraid, with which it is possible to seamlessly assemble multiple reusable gene modules, including promoters, genes and terminators, together in a binary vector in a single restriction-ligation. even though a lot of cloning systems are now available as discussed above, many common problems still exist in our routine experiment. in modern functional genomic study, one of challenges is to construct a set of vectors for a gene of interest to study gene functions in special spatio-temporal patterns. it includes cloning rapidly and efficiently, labeling proteins with different tags, shuffling randomly promoters and genes, introducing a desired regulatory element for a given promoter or gene, and modifying vectors in hand for ongoing demands. therefore, the flexibility of vectors appears to be a main challenge. what’s more, gateway technology available now leaves a common question to be answered: after lr reaction, there is a longer spacer existed between genes and tags, which may result in detrimental effect on protein functions. in this study, we developed a highly flexible expression system to cope with on-going demands in plant functional genomic study, based on classical cloning approaches other than to introduce a new method. firstly, we employed widely-used site-specific recombination cloning system for high cloning efficiency. secondly, we introduced two pairs of ssrs, one for genes and the other for promoters in two independent entry clones. thirdly, we adopted traditional restriction enzyme/ligase strategy in an easy-operating intermediate cloning vector, instead of a difficult-operating binary vector as previous researchers used. this rel is designed for cloning of desired multiple regulatory elements or tags, and it also provide flexible choices for cloning.  through directly synthetizing sequences of all dna fragments except the backbone of vectors indicated below, we developed our cloning system, biovector. biovector is composed of three basic vectors: a gene entry clone with one pair of ssrs of attl1/attl a promoter entry clone with another pair of ssrs of attl3/attl and a binary destination vector with two pairs of corresponding ssrs of attr1/attr and attr3/attr therefore, with a single lr reaction, a promoter can be fused to a gene and a plant expression vector is constructed. gene entry clone the gene entry clone was developed from the puc vector due to its advantage of easy-operating for cloning, and introduced up to restriction enzyme/ligase sites including t/a cloning sites, therefore it is convenient to clone any dna fragments and to assemble multiple dna fragments into a gec. rel sites were inserted inside site-specific recombination sites in gecs, so that it was possible to make a seamless fusion between a gene and a tag in our system, rather than to introduce a ssr spacer between them as widely-used gateway cloning system does, in which the tags are in destination vectors. such a spacer has the potential to result in the synthesis of a non-functional or insoluble protein-tag fusion. another improvement was that a stop code was put at the end of rel sites and inside ssr. thus, only the gene without a stop code was needed to be cloned into a gec, then it can be removed from one gec to another by a simple rel. in this case, there was no need to clone a gene for the n- or c-terminal fusion, respectively, as wide-used gateway cloning systems do. therefore, gecs saved both time and cost for cloning and sequencing of genes. ✶all vectors were delivered to abrc. “n” or “c” indicates that the position of a tag is at the “n” or “c” terminus of a target gene, “n” or “c” for the “n” or “c” half part of yfp fluorescence protein. all vectors contain recombination sites of attl and attl and have the chloramphenicol selection marker in e. coli. all the sequences are showed in additional file therefore, genes, tags, and any regulatory elements of interest from pcr amplification can be directly cloned into gecs after digestion. we constructed a set of alternative gecs for labeling a desired protein with different tags at the n- or c-terminus. and all gecs shared identical rel sites, and it was easy to move a gene from one gec to another with a simple rel. a ccdb gene in rel sites served as a negative selection marker for e. coli dh5α as all gateway entry vectors employ. the chloramphenicol was employed as a selection marker in e. coli, which may be compatible to most of binary vectors available. we designed a primer entry-f for forward sequencing, and the general primer sp can be used as a reverse sequencing primer. plenty of rel sites in gecs provide a flexible platform for ad arbitrium modifying the vector by researcher themselves for their own individual study. this is an important respect of this flexible system, because the plasticity of a system is always demanded in functional genome study, but a shortage of wide-used gateway systems. for example, to insert rna-binding loops inside ssrs in gec makes it possible to label an rna. one of our considerations was to develop gecs compatible with the present gateway destination vectors for plants, e. coli, and yeast, so researchers can keep their vectors in hands when introduce biovector. promoter entry clone most of gateway based cloning systems normally put a constitutive promoter, such as camv 35s, in the binary vector, in which the promoter is not easy to be replaced by another promoter due to its low cloning efficient. even though multiple gateway system clone a promoter inside the specific ssr sites, it bears low efficient in subsequent multiple recombination reactions and it is also difficult to insert a cis-element next to the promoter. the promoter entry clone in biovector also originated from puc vector, shared the same backbone as gec, and embraced up to rel sites inside ssr sites. the plenty of rel sites benefited to freely clone or assemble various promoters or cis-elements together to study their functions. obviously, as in gecs, pecs were good to be updated by researchers for an ambitious idea. there were three pecs available with no enhancer, one enhancer, or two enhancers, respectively. thus, a gene can be expressed or over-expressed from a native promoter, and the bona fide functions of a gene could be revealed. such a strategy was designed to avoid disadvantages of generally over-expressing promoter camv 35s, which has overt ectopic effects, weak or null functions in some tissues and plants, and adverse effects on adjacent genes. ✶ all vectors were delivered to abrc. all vectors contain recombination sites of attl and attl and have the chloramphenicol selection marker in e. coli. all the sequences are showed in additional file again, the three pecs had identical rel sites, which conferred to easily shift promoters among them. the selection marker and the sequencing primers in pecs were same to that of gecs. besides rel strategy, other cloning technology, such as in-fusion® hd cloning from clontech, can be an alternative efficient approach for cloning genes or promoters into gecs or pecs, so as to increase the cloning efficiency. if introducing the type iis endonucleases, such as bsa i, bbs i, and bsmb i, gecs and pecs could be compatible to golden gate cloning and goldenbraid. binary destination vector all binary destination vectors in this study were developed from pgreen/pclean, which are highly efficient in a wide arrange of plants. so, bdvs have smaller size than most of gateway-compatible binary vectors. and the small size of binary vectors facilitates to increase the cloning efficiency, plasmid yield and plant transformation. because the right border of the t-dna is largely preserved whereas the left border is frequently truncated after integration, a selectable marker in planta was placed next to the left border in bdvs to ensure that all transformants with a positive marker always carry the introduced gene. a set of binary vectors were designed for a genomic gene or cds gene expression, gene silencing, and ethanol-inducible expression, respectively. there were more bdvs available with various selection markers in e. coli and plants, providing multiple choices in different projects. so, biovector can be used to express a genomic gene spanning the sequence from the promoter to the terminator, to analyze the function of coding sequence from a desired promoter or ethanol-inducible promoter, to monitor proteins with fluorescent and other tags, to study protein-protein interaction, and to silence a gene in specific spatio-temporal mode. the mcs in bdvs facilitates to be modified for extensive demands, such as replacing the preloaded transcription terminator or the selection marker. e. coli ✶all vectors were delivered to abrc. all the sequences are showed in additional file once a gene and a promoter were cloned into gecs or pecs, respectively, the gene and the promoter can be assembled into a binary vector through a single lr reaction, which is a high efficient and directional reaction. several systems, such as multisite gateway cloning, provide a possibility to combine promoters and genes at will, but with limited numbers of promoters and tags available. biovector, however, made it easy to express a gene from different desired promoters with different intensities. gene entry clones from wide-used gateway system made by most researchers shared the same ssr sites attl1/ at both ends with gecs. thus, gene entry clones at hand were compatible with our bdvs, rather than discarding them when introduce biovector. obviously, gecs and pces can serve as an intermediate vector for cloning due to plenty of mcss available. in addition, gecs, pecs and bdvs can be shared worldwide as a library. we have delivered all vectors in this study to abrc centre. verification of biovector with biovector we succeeded in stable or transient expression of gfp with or without nuclear localization signal or nuclear export signal in arabidopsis protoplasts, a gus gene from the companion cell-specific sucrose h+symporter promoter with or without enhancers in arabidopsis, a myc-tagged gene in nicotiana benthamiana, and a luciferase gene under ethanol-inducible pattern n. benthamiana. the results supported that biovector was efficient expression vector for plants.  perfect combination of conventional rel strategy and modern ssr technology confers obvious advantages to biovector. exchangeable and efficient. a gene and a promoter can be easily assembled together to fulfill expressing a gene from a temporal-spatio promoter with different intensity, especially overexpressing genes under the control of native promoters; flexible. gecs, pecs, and bdvs can be ad arbitrium modified with ongoing demands; practical and versatile. biovector can be applied to almost all fields in functional genome research of various plants; universal and time-/labour-saving. gecs can be efficiently applied to any plant, yeast, and e. coli destination vectors sharing corresponding ssr sites, and it is possible to construct a worldwide library as shared community resource for gecs, pecs and bdvs; seamless fusion. it is possible to make seamless fusion between a protein and a tag, rather than to introduce a detrimental ssr spacer as the widely-used gateway recombination system does; broad application and interest. the idea of biovector can also be applied to similar study in animals and yeast. 
to improve efficiency and flexibility for constructing expression vectors, we developed a platform, biovector, by combining classical restriction enzyme/ligase strategy with modern gateway dna recombination system. functional genomic research always needs to assemble different dna fragments into a binary vector, so as to express genes with different tags from various promoters with different levels. the cloning systems available bear similar disadvantages, such as promoters/tags are fixed on a binary vector, which is generally with low cloning efficiency and limited for cloning sites if a novel promoter/tag is in need. this system included a series of vectors for gene cloning, promoter cloning, and binary vector construction to meet various needs for plant functional genomic study. this biovector platform makes it easy to construct any vectors to express a target gene from a specific promoter with desired intensity, and it is also waiting to be freely modified by researchers themselves for ongoing demands. another disadvantage is that a long spacer from recombination sites, which may be detrimental to the protein function, exists between a gene and a tag.
 functional genomic research always needs to assemble different dna fragments into a binary vector, so as to express genes with different tags from various promoters with different levels. the cloning systems available bear similar disadvantages, such as promoters/tags are fixed on a binary vector, which is generally with low cloning efficiency and limited for cloning sites if a novel promoter/tag is in need. therefore, it is difficult both to assemble a gene and a promoter together and to modify the vectors in hand. another disadvantage is that a long spacer from recombination sites, which may be detrimental to the protein function, exists between a gene and a tag. multiple gateway system only resolves former problem at the expense of very low efficiency and expensive for multiple lr reaction.  to improve efficiency and flexibility for constructing expression vectors, we developed a platform, biovector, by combining classical restriction enzyme/ligase strategy with modern gateway dna recombination system. this system included a series of vectors for gene cloning, promoter cloning, and binary vector construction to meet various needs for plant functional genomic study.  this biovector platform makes it easy to construct any vectors to express a target gene from a specific promoter with desired intensity, and it is also waiting to be freely modified by researchers themselves for ongoing demands. this idea can also be transferred to the different fields including animal or yeast study. expression vectorgene specific-expressionfunctional genome studygene cloningrestriction enzyme/ligase strategygateway dna recombination 