Subcloning Synthetic ORF Sequences Into pENTR223.1-Sfi  and pDONR223.1

 

Synthetic protein-coding sequences (ORFs) were prepared, under contract to the Mammalian Gene Collection (MGC; (1)). In most cases the synthetic ORF sequence exactly matched the assigned RefSeq mRNA sequence. In a minority of cases isocodon changes were introduced into the ORF (to facilitate its synthesis and subcloning), and these changes are annotated in the GenBank record. The ORF sequence was confirmed by DNA sequence analysis to MGC standards for sequence quality.   

 

The ORF sequence was prepared with additional flanking DNA sequence to facilitate its subcloning into either of two Gateway™ cloning system (2, 3) vectors (provided by Invitrogen Corp.). One vector, pENTR223.1-Sfi permits directional subcloning of the insert using restriction enzymes and ligase.  The second vector, pDONR223.1 allows recombinational subcloning of the ORF sequence, mediated by BP Clonase.  Both approaches yield an identical subclone of the ORF in pENTR223.1.

 

ORFs subcloned using restriction enzymes and ligase are inserted between two Sfi I sites, situated between the attL1 and attL2 sites of pENTR223.1-Sfi. Non-identical, three-nucleotide (nt) 3’ overhangs created by Sfi I cleavage of pENTR223.1-Sfi permit directional subcloning. (A few of the subclones were prepared using pENTR223.1 together with the Infusion™ Cloning system (4); these give identical products to those prepared with restriction enzymes and ligase.)

 

Alternatively, ORFs were synthesized with flanking attB1 and attB2 recombination sequences, allowing them to be subcloned by site-specific recombination, using BP Clonase, into pDONR223.1 to yield subclones in pENTR223.1.  Additional detail on this reaction is provided below.

 

ORFs with stop codons use the naturally occurring stop codon.  Other ORFs were designed with the stop codon removed, to allow synthesis of C-terminal fusions with proteins encoded by an appropriately designed expression vector (Gateway Destination vector).   

 

Each ORF subcloned into pENTR223.1-Sfi is designed to have the proper reading frame of the ORF in phase with the triplets at the proximal end of attL1 (underlined) and the adjacent Sfi I sequence, thus:

 

5’...AAA AAA GCA GAA GGG CCG  TCA AGG CCC acc ATG–5’ORF3’-

 

The ORF-proximal end of the attL1 site is underlined, and the Sfi I site is in light grey (with the 3-nt 3’ overhang in red).  Following the Sfi I site is a minimal Kozak acc consensus, in lower case, and the initiating ATG of the ORF, in capital letters.

 

At the 3’ end of the ORF, the junction with the flanking sequences (and the vector) likewise maintains the phase of reading frame, with the triplets indicated in light grey within the Sfi I site (with the 3-nt 3’overhang in dark grey) and the proximal end of attL2 (underlined), thus:

 

-5’ORF3’- GGC CTC ATG GGC CCA GCT TTC TTG . . .3’

 

Subcloning by site-specific recombination using pDONR223.1

Synthetic ORFs were designed to be substrates for attB x attP recombination, using BP Clonase, to transfer the ORF into the pDONR223.1 vector to create an Entry clone in the pENTR223.1 vector.  The reaction mediated by BP Clonase is:

 

pDONR223.1 (SpnR) + attB1-ORF-attB2 à pENTR223.1-ORF (SpnR) + attR(CmR+ccdB) byproduct.  (Here attB1-ORF-attB2 is a synthetic DNA.)

 

This reaction is analogous to BxP cloning of attB-PCR products. The products were transformed into T-phage-resistant E. coli competent cells and SpnR colonies selected on spectinomycin-containing plates.

 

The synthetic attB1-ORF-attB2 has the following structure:

 

Additional details on the cloning of attB-PCR products and the BP Clonase reaction conditions are available on the Invitrogen web site at:  https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=561&

 

 

Map of Vector pENTR223.1-Sfi (Invitrogen Custom Gateway Entry Vector)

 

This vector carries two genes in the region between the two Sfi I sites (ccdB and CmR) that will be replaced by cloned DNA segments.  The ccdB gene (DNA gyrase inhibitor) provides strong negative selection against vector molecules retaining this region, whereas CmR (chloramphenicol resistance gene) provides positive selection for propagating the vector. Because ccdB is toxic to most standard strains of E. coli, it is important to propagate this vector in E. coli DB3.1 (gyrA) or in ccdBSurvival competent cells (ccdB-resistant; T1 & T5 phage resistant), both available from Invitrogen.

 

Sequencing primers (M13F and T7 Rev) that prime from outside of the attL sites are generally suitable for sequencing inserts larger than ~500 nt, but they may provide incomplete sequences for smaller inserts, due to L1-L2 hairpin formation (5). The use of sequencing primers GW1 and GW2 should be suitable for sequencing of all sizes of inserts.

 

Molecule Features:

Start     End   Name         

 

   1     534   ori         

1113    1149   fwd Seq primers (includes M13F: 1113-1129) 

1264    1165 C attL1  

1203    1237   GW1 

1617    1312 C ccdB        

2618    1959 C CmR         

2786    2885   attL2

2823    2847 C GW2      

2943    2902 C rev Seq primers (includes T7Rev: 2903-2922) 

3154    4164   SpnR

 

 

Map of Vector pDONR223.1 (Invitrogen Custom Gateway Entry Vector)

This vector carries two genes in the region between the two attP sites (ccdB and SpnR) that will be replaced by cloned DNA segments.  This vector must be propagated in E. coli DB3.1 (gyrA) or in ccdBSurvival competent cells (ccdB-resistant; T1 & T5 phage resistant).

 

 

Molecule:       pDONR223.1, 5005 bps DNA Circular

 

Description:    Custom Vector from Invitrogen

 

Molecule Features:

 

Type     Start     End   Name         Description

 

MARKER     534           Ori         

GENE       840    1106   T1-T2       

REGION    1113    1149   M13f        

GENE      1166    1364   attP1       

REGION    1203    1227   GW1          Forward seq primer

GENE      1793    2092   ccdB        

GENE      2440    3099   CmR         

REGION    3541    3517 C GW2          Reverse seq primer

GENE      3578    3380 C attP2       

REGION    3637    3596 C M13r        

GENE      3848    4858   SpnR        

 

 

References:

1.         MGC-NIH (2006) http://mgc.nci.nih.gov/.

2.         Hartley, J.L., Temple, G.F. and Brasch, M.A. (2000) DNA cloning using in vitro site-specific recombination. Genome Res, 10, 1788-95.

3.         https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewCategories&pc=110&npc=92&nc=109&.

4.         http://www.clontech.com/clontech/whatsnew/announcements/9_26_05_infusion_v2.shtml, C.

5.         Esposito, D., Gillette, W.K. and Hartley, J.L. (2003) Blocking oligonucleotides improve sequencing through inverted repeats. Biotechniques, 35, 914-6, 918, 920.