Integrated DNA Technologies - Custom Gene Synthesis

Custom Gene Synthesis

Documentation and Support

FAQs

IDT offers a confidential custom gene synthesis service. By ordering genes from IDT, researchers not only save money spent on reagents necessary for construction, cloning, and sequencing but can also save time by outsourcing the manufacturing of hard-to-clone gene sequences which often result in repeated failures. At IDT, all genes are constructed using Ultramers™, the highest fidelity next generation synthesis technology available. Genes arrive in a plasmid cloning vector and are ready for use in a variety of applications.

Sequence information is always secure and confidential at IDT. Non-disclosure agreements are available through IDT’s legal services upon request.

OrderingOverviewVectorsProtocolsBiosecurity

Product Configuration

	miniGENES (up to 400 bp)	Genes (401 + bp)
Pricing	$220.00 USD	Starting at $0.55 USD / bp
Turnaround Time	4-8 business days	8-12 business days

*The time it takes to make a gene is dependent on length, complexity, and vector choice.

*Extra charges may apply for gene segments with added complexity (such as homopolymeric runs, critical hairpin structures or G/C content), which can interfere with assembly and/or sequencing performance. For more information about potential types of complexity, please see the Overview section below.

Place an order now

Genes ordered from IDT will go through the following process:

Design review and consultation

Researchers enter the gene sequence on the gene entry site.
A/C/G/T bases only

no mixed degenerate bases
no “modified" bases (Inosine, Uridine)

The sequence is automatically analyzed for the following characteristics which may interfere with synthesis, assembly, or sequencing performance:

Hairpins: Hairpins >10 bases and G/C-rich secondary structures
Repeats and homo-polymeric runs: long repeats, >10 bases for G/C, or >30 bases for A/T
Overall and regional GC content: content >65% and <25% for G/C
Restriction Site Analysis: restriction site duplications and Dam/Dcm Methylation sites

Dam Methylation Site = G^mATC
Dcm Methylation Site = C^mCWGG

If the sequence does not pass the screen criteria, you will be contacted by a gene services specialist to determine the best way to proceed.
The sequence will also be analyzed to ensure safety and regulatory conformance.

A biohazard disclosure statement is required for each gene order.
IDT reserves the right to refuse any order that does not pass this analysis.

Codon Optimization

Different organisms vary in the frequency with which they use certain codons and exhibit varying degrees of codon preferences. The fastest growing microbes tend to have the highest codon biases. This codon bias is thought to be reflective of the abundance of the corresponding tRNA pool. Gene sequences containing preferred codons for the expression host are translated more efficiently, with greater accuracy, and in greater yield. In addition, optimization can be used to disrupt difficult sequence features (such GC content, hairpins, repeats, etc.). A codon optimization tool is available on the IDT website.

Sequence verification

Once a gene is cloned, IDT verifies the sequence with double-stranded DNA sequencing. We also provide the sequencing chromatograms and a plasmid map to the customer.

Guaranteed yields

IDT will provide 2 µg of purified double-stranded DNA in a circularized plasmid, delivered lyophilized.

Confidentiality

All sequence information is confidential at IDT
All online ordering steps, including sequence entry and choice of parameters, is secure and protected.

Gene Synthesis Applications

IDT provides the gene in a cloning vector so that it is amendable to further subcloning into a vector of the researcher’s choice. These genes can then be used in a number of applications including, but not limited to:

Protein Expression

Recombinant antibodies
Novel fusion proteins
Codon optimized short proteins
Functional peptides: catalytic, regulatory, binding domains

microRNA genes
Template for in vitro transcription (IVT)
shRNA expression cassettes
Regulatory sequence cassettes
Synthetic repeat constructs
Synthetic chromosomes/genomes
Custom vectors
Microarray-ready cDNA
Gene variants and SNPs
DNA vaccines and vectors
Standards for quantitative PCR and other assays
Functional Genomics

Custom genes offer near limitless flexibility for protein mutagenesis

Unrestricted point mutations
Mutant libraries
Large deletion mutants

Standard Cloning

IDT has the ability to clone in-house in a range from 50bases -5.0kb. All genes, unless otherwise specified, will be delivered in IDT’s proprietary cloning vector, pIDTSmart. This vector has been specifically engineered to remove most common restriction endonuclease cleaving sites and does not contain a promoter within the cloning region. If you require restriction sites or promoters for your application, it is important to include these when ordering the gene of interest by appending the desired restriction sites or promoters to the ends of your gene construct. The default version of pIDTSmart contains a kanamycin resistance cassette. An ampicillin version is also available upon request.

Custom Vector Requests

IDT has the ability to sub-clone into almost any customer supplied vector. To get the process started, go to IDT’s website (www.idtdna.com/order/customvector.aspx) and complete the Custom Vector request sheet or contact IDT Gene Support at genes@idtdna.com. An IDT representative will contact you within 2 business days to discuss your custom vector request.

Vector

Endogenous Enzymes

Sequence

# of Cuts

Comments

pIDTBlue

ApaLI

GTGCAC

BspDI

ATCGAT

ClaI

ATCGAT

EagI

CGGCCG

Doesn't Exist in <= 400 version

PvuI

CGATCG

PvuII

CAGCTG

RsaI

GTAC

Restriction Sites not Present in Plasmid

Acc65I, AgeI, ApaI, AscI, Asp718I, AvaI, AvrII, BamHI, BglII, BmgBI, BstBI, DraII, Ecl136II, EcoRI, EcoRV, HindIII, HpaI, KpnI, MluI, NcoI, NdeI, NheI, NotI, NsiI, PacI, PaeR7I, PspOMI, PstI, SacI, SacII, SalI, SmaI, SnaBI, SpeI, SphI, TliI, TspMI, XbaI, XhoI, XmaI

Vector

Endogenous Enzymes

Sequence

# of Cuts

Comments

pIDTSmart-KAN/AMP

ApaL

GGGCCC

Doesn't Exist in > 400 version or pIDTSMART-KAN

ApaLI

GTGCAC

Contains 1 in pIDTSMART-KAN

BspDI

ATCGAT

Contains 2 in pIDTSMART-KAN

ClaI

ATCGAT

Contains 2 in pIDTSMART-KAN

DraII

RGGNCCY

Contains 1 in pIDTSMART-KAN

NsiI

ATGCAT

Doesn't Exist in <= 400 version

PspOMI

GGGCCC

Doesn't Exist in > 400 version or pIDTSMART-KAN

PvuI

CGATCG

RsaI

GTAC

SnaBI

TACGTA

Restriction Sites not Present in Plasmid

Acc65I, AgeI, AscI, Asp718I, AvaI, AvrII, BamHI, BglII, BmgBI, BstBI, EagI, Ecl136II, EcoRI, EcoRV, HindIII, HpaI, KpnI, MluI, NcoI, NdeI, NheI, NotI, PacI, PaeR7I, PstI, PvuII, SacI, SacII, SalI, SmaI, SpeI, SphI, TliI, TspMI, XbaI, XhoI, XmaI

Resuspension

Centrifuge the tube prior to opening to ensure that the DNA is at the bottom of the tube.
Resuspend DNA in 20 µL of 10 mM Tris, 0.1 mM EDTA buffer (pH 7.5 – 8.0) to reach an approximate concentration of 0.1 µg/µL for a stock concentration.
Incubate the tube at room temperature for 30 minutes and then vortex for 20 seconds
Centrifuge for 1 minute
To create a working concentration, add 1 µL of the stock concentration to 999 µL of water to reach an approximate working concentration of 0.1 ng/µL. Use 1-2 µL of the working dilution for bacterial transformations.

Storage Conditions

Store hydrated DNA at -20°C.

Lyophilized DNA is stable at room temperature for 3 months. We recommend storing at -20°C for longer periods of time.

Transformations (Chemical)

Place frozen competent cells and pre-labeled tubes on ice
Pre-warm the hot bath to 42°C
Once cells are thawed on ice, aliquot 25 µL of XL1 Blue cells to each of the pre-chilled tubes. Keep all the tubes on ice
Add 2 µL of ligation or PCR reaction to cells and gently swirl the cells with pipette tip
Incubate on ice for 30 minutes
Heat shock tubes for 45 seconds in 42°C hot bath
Return tubes to ice for 2 minutes
Add 250 µL of SOC Media to each tube and place tubes in a 37°C shaking incubator for 1 hour
Pre-warm agar plates to 37°C in incubator
Spread 125 µL of cells on agar plates
After spreading cells, let plates sit for approximately 10 minutes at room temperature. Then place the plates upside down in 37° incubator for 12 – 24 hours

Inoculation

For a large number of colonies, use a 2.2 mL deep well plate and add 1.6 mL of LB Broth with the appropriate antibiotic.
For a small number of colonies, use a 14 mL round bottom culture tube and add 2 mL of LB Broth with the appropriate antibiotic
Touch the colony with a toothpick or pipette tip and place it in the broth
Cover with a gas permeable lid and place in a 37° shaking incubator for 12 – 20 hours.

Digestions

The standard in-house digestion protocol is below:

Total Volume	50µL
DNA	400ng
Buffer	5µL
Water	XµL
Enzyme(s)	1µL each

Incubate for 1 hour at the temperature recommended by the enzyme manufacturer. Take 2-5 µL of the digested sample, add loading dye, and run on a gel to verify that the digestion took place. In separate lanes, also run a ladder to verify the size of the product and a sample of undigested product as a control.

*The length of running time for the gel will depend on the size of the digested insert. Smaller inserts require shorter run times or the product will run off the gel.

*The enzyme should not be more than 1/10^th of total reaction

Reagents

Material	Components		Amount (g or ml)/L
STE Buffer pH 7.5	10 mM	Tris	1.21
	10 mM	NaCl	2.92
	1 mM	EDTA	0.37
10X Taq Ligase Buffer pH 7.6	200 mM	Tris	24.22
	250 mM	Potassium Acetate	24.54
	100 mM	Magnesium Acetate (tetrahydrate)	21.45
	100 mM	DTT	15.42
	100 mM	NAD	6.85
	1 %	Triton-X100	24.22
1X T4 DNA Ligase Buffer pH 7.5	50 mM	Tris	6.05
	10 mM	Magnesium Acetate (tetrahydrate)	2.03
	10 mM	DTT	1.54
	1 mM	Adenosine 5′-triphosphate disodium salt	0.55
	25 µg/ml	BSA	0.025
Agar Plates with Amp	1 %	Tryptone	10
	1 %	Sodium Chloride	10
	0.5 %	Yeast Extract	5
	1.5 %	Agar	15
	100 µg/ml	Ampicillin	0.1
LB Broth with Amp	1 %	Tryptone	10
	1 %	Sodium Chloride	10
	0.5 %	Yeast Extract	5
	100 µg/ml	Ampicillin	0.1
Agar Plates with Kan	1 %	Tryptone	10
	1 %	Sodium Chloride	10
	0.5 %	Yeast Extract	5
	1.5 %	Agar	15
	50 µg/ml	Kanamycin	0.05
LB Broth with Kan	1 %	Tryptone	10
	1 %	Sodium Chloride	10
	0.5 %	Yeast Extract	5
	50 µg/ml	Kanamycin	0.05
SOC Media pH 7.5	2 %	Tryptone	20
	0.5 %	Yeast Extract	5
	0.05 %	Sodium Chloride	0.5
	2.5 mM	Potassium Chloride	0.19
	10 mM	Magnesium Chloride (hexahydrate)	20.3
1X Loading Dye	0.4 %	Bromophenol Blue	4
	0.4 %	Xylene Cyanol FF	4
	50 %	Glycerol	500

Gene Biosecurity

As one of five founding members of the International Gene Synthesis Consortium (IGSC), IDT’s gene sequence and customer screening practices are consistent with the IGSC’s Harmonized Screening Protocol. To prevent the misuse of synthetic genes, IDT screens the sequences of ordered genes to identify regulated and other potentially dangerous pathogen sequences, and verifies that its customers are legitimate scientists engaged in beneficial research.

For more information about the IGSC and the Harmonized Screening Protocol, please visit the website at http://www.genesynthesisconsortium.org/Home.html.

The United States government has published draft guidance describing how commercial providers of synthetic genes should perform gene sequence and customer screening. IDT, along with the other IGSC member companies, supports the adoption of that guidance in substance and will implement the procedures that the final guidance recommends. For more information, please see http://edocket.access.gpo.gov/2009/E9-28328.htm.