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Brian Naughton | Sat 16 August 2014 | biotech | DNA vaccine

Starting with a protein sequence, there are several ways to produce a DNA sequence with codons that are optimized for expression. I created a DNA sequence for my hybrid flagellin using Life Tech GeneOptimizer. I selected Mus musculus as the target organism and I also selected the option to add a Kozak sequence. Here's what gets spit out.

GCCACCATGGCCCAAGTGATCAACACCAACAGCCTGAGCCTGATCACCCAGAACAACATCAACAAGAACCAGAGCGCCCTGAGCAGCAGCATCGAGAGAC
TGAGCAGCGGCCTGAGAATCAACAGCGCCAAGGACGATGCCGCTGGCCAGGCTATCGCCAACAGATTCACCAGCAACATCAAGGGCCTGACCCAGGCCGC
CAGAAACGCCAACGATGGCATCAGCGTGGCCCAGACAACAGAGGGCGCACTGAGCGAGATCAACAACAACCTGCAGAGAGTGCGCGAGCTGACCGTGCAG
GCCACCACAGGCACAAACAGCGAGAGCGACCTGTCCAGCATCCAGGACGAGATCAAGAGCAGACTGGATGAGATCGACAGAGTGTCCGGCCAGACCCAGT
TCAACGGCGTGAACGTGCTGGCCAAGAACGGCAGCATGAAGATCCAAGTGGGCGCCAACGACAACCAGACCATCACCATCGACCTGAAGCAGATCGACGC
CAAGACCCTGGGCCTGGACGGCTTCTCTGTGAAGGCCCTGATCACAGCCAGCGGCGACATCAGCCTGACCTTCAAACAGGTGGACGGCGTGAACGACGTG
ACCCTGGAAAGCGTGAAGGTGTCCAGCTCTGCCGGCACAGGCATCGGCGTGCTGGCTGAAGTGATTAACAAGAACAGCAACAGGACCGGCGTGAAGGCCT
ACGCCAGCGTGATCACCACAAGCGACGTGGCCGTGCAGAGCGGCAGCCTGTCTAACCTGACCCTGAACGGCATCCACCTGGGCAATATCGCCGATATCAA
GAAGAACGACAGCGACGGCAGACTGGTGGCCGCCATCAACGCCGTGACAAGCGAGACAGGCGTGGAAGCCTACACCGACCAGAAGGGCAGACTGAACCTG
AGATCCATCGACGGCAGGGGCATCGAAATCAAGACCGACAGCGTGTCCAACGGCCCCTCCGCCCTGACAATGGTCAACGGCGGACAGGACCTGACCAAGG
GCAGCACCAACTACGGCAGGCTGTCCCTGACAAGACTGGACGCCAAGTCCATCAACGTGGTGTCCGCCAGCGACTCCCAGCACCTGGGCTTTACAGCTAT
CGGCTTCGGCGAGAGCCAGGTGGCCGAGACTACCGTGAATCTGAGGGACGTGACCGGCAACTTCAACGCTAACGTGAAGTCCGCCTCCGGCGCTAACTAC
AACGCTGTGATCGCCTCTGGCAACCAGAGCCTGGGAAGCGGCGTGACCACACTGAGAGCTGTGGCCAACGGCAAGACCACCGACCCCCTGAAGGCTCTGG
ACGACGCTATCGCCTCCGTGGACAAGTTCAGATCCAGCCTGGGCGCTGTGCAGAACAGACTGGACAGCGCCGTGACCAACCTGAACAACACCACCACCAA
CCTGTCCGAGGCCCAGAGCAGAATCCAGGATGCCGACTACGCCACCGAGGTGTCCAACATGAGCAAGGCCCAGATCATCCAGCAGGCCGGCAACTCTGTG
CTGGCTAAGGCCAACCAGGTGCCCCAGCAGGTGCTGAGTCTGCTGCAGGGA

When I align my sequence with the original BioBrick created for the Slovenian iGem project, the two DNA sequences are only about 75% concordant (the protein sequences were identical). I expected some differences but not this many. My best guess is that the Slovenian BioBrick is not intended for use in mouse, but I really don't know what is going on. It would be interesting to test the difference in expression between the two sequences in a mouse.

CLUSTAL O(121) multiple sequence alignment

LifeTech              GCCACCATGGCCCAAGTGATCAACACCAACAGCCTGAGCCTGATCACCCAGAACAACATC
Slovenia              ------atggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatc
                            ***** ***** ** ** ***********    ******** ** ** ** ***

LifeTech              AACAAGAACCAGAGCGCCCTGAGCAGCAGCATCGAGAGACTGAGCAGCGGCCTGAGAATC
Slovenia              aacaagaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtatt
                      ************   ** ***   **    ****** * ***      *** ** * **

LifeTech              AACAGCGCCAAGGACGATGCCGCTGGCCAGGCTATCGCCAACAGATTCACCAGCAACATC
Slovenia              aacagcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacatt
                      ******** ***** ** ** ** ** ***** ** ** *** * ******   *****

LifeTech              AAGGGCCTGACCCAGGCCGCCAGAAACGCCAACGATGGCATCAGCGTGGCCCAGACAACA
Slovenia              aaaggcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccacc
                      ** ******** ***** *** * *********** ** ***  *** ** ***** **

LifeTech              GAGGGCGCACTGAGCGAGATCAACAACAACCTGCAGAGAGTGCGCGAGCTGACCGTGCAG
Slovenia              gaaggcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacag
                      ** ***** ***  *** ************ * *** * ***** ** ***** ** ***

LifeTech              GCCACCACAGGCACAAACAGCGAGAGCGACCTGTCCAGCATCCAGGACGAGATCAAGAGC
Slovenia              gccactaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcc
                      ***** ** ** ** ***   ***   ** *****    *********** ** **   *

LifeTech              AGACTGGATGAGATCGACAGAGTGTCCGGCCAGACCCAGTTCAACGGCGTGAACGTGCTG
Slovenia              cgtctggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctg
                      * ******** ** *** * ** ** ** ******************************

LifeTech              GCCAAGAACGGCAGCATGAAGATCCAAGTGGGCGCCAACGACAACCAGACCATCACCATC
Slovenia              gcaaaaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatc
                      ** ** ** ***  ****** ***** ** ***** ** ** ******** ***** ***

LifeTech              GACCTGAAGCAGATCGACGCCAAGACCCTGGGCCTGGACGGCTTCTCTGTGAAGGCCCTG
Slovenia              gatctgaagcagattgatgctaaaactcttggccttgatggttttagcgttaaagcgtta
                      ** *********** ** ** ** ** ** ***** ** ** **    ** ** **  *

LifeTech              ATCACAGCCAGCGGCGACATCAGCCTGACCTTCAAACAGGTGGACGGCGTGAACGACGTG
Slovenia              atcacggcttctggggatattagcttgacttttaaacaagtggatggcgtgaatgatgta
                      ***** **    ** ** ** *** **** ** ***** ***** ******** ** **

LifeTech              ACCCTGGAAAGCGTGAAGGTGTCCAGCTCTGCCGGCACAGGCATCGGCGTGCTGGCTGAA
Slovenia              actttagagagcgtaaaagtttctagttcagcaggcacggggatcggtgtgttagcggaa
                      **  * ** ***** ** ** ** ** ** ** ***** ** ***** *** * ** ***

LifeTech              GTGATTAACAAGAACAGCAACAGGACCGGCGTGAAGGCCTACGCCAGCGTGATCACCACA
Slovenia              gtgattaacaaaaattctaaccgaacaggggttaaagcttatgcgagcgttatcaccacg
                      *********** **    *** * ** ** ** ** ** ** ** ***** ********

LifeTech              AGCGACGTGGCCGTGCAGAGCGGCAGCCTGTCTAACCTGACCCTGAACGGCATCCACCTG
Slovenia              agcgatgtggcggtccaatcaggaagtttgagtaatttaactttaaatgggatccatttg
                      ***** ***** ** **    ** **  **  ***  * **  * ** ** *****  **

LifeTech              GGCAATATCGCCGATATCAAGAAGAACGACAGCGACGGCAGACTGGTGGCCGCCATCAAC
Slovenia              ggtaatatcgcagatattaagaaaaatgactcagacggaaggttagtcgcagcgatcaat
                      ** ******** ***** ***** ** ***   ***** **  * ** ** ** *****

LifeTech              GCCGTGACAAGCGAGACAGGCGTGGAAGCCTACACCGACCAGAAGGGCAGACTGAACCTG
Slovenia              gcggttacttcagaaaccggcgtggaagcttatacggatcaaaaagggcgcttgaatttg
                      ** ** **    ** ** *********** ** ** ** ** ** **  *  ****  **

LifeTech              AGATCCATCGACGGCAGGGGCATCGAAATCAAGACCGACAGCGTGTCCAACGGCCCCTCC
Slovenia              cgcagtatagatggtcgtgggattgaaatcaaaaccgatagcgtcagtaatgggcctagt
                      *    ** ** **  * ** ** ******** ***** *****    ** ** **

LifeTech              GCCCTGACAATGGTCAACGGCGGACAGGACCTGACCAAGGGCAGCACCAACTACGGCAGG
Slovenia              gctttaacgatggtcaatggcggtcaggatttaacaaaaggttctactaactatgggagg
                      **  * ** ******** ***** *****  * ** ** **    ** ***** ** ***

LifeTech              CTGTCCCTGACAAGACTGGACGCCAAGTCCATCAACGTGGTGTCCGCCAGCGACTCCCAG
Slovenia              ctttctctcacacgcttagacgctaaaagcatcaatgtcgtttcggcttctgattcgcaa
                      ** ** ** *** *  * ***** **   ****** ** ** ** **    ** ** **

LifeTech              CACCTGGGCTTTACAGCTATCGGCTTCGGCGAGAGCCAGGTGGCCGAGACTACCGTGAAT
Slovenia              catttaggtttcacagcgattggttttggggaatctcaagtggcagaaaccacggtgaat
                      **  * ** ** ***** ** ** ** ** **    ** ***** ** ** ** ******

LifeTech              CTGAGGGACGTGACCGGCAACTTCAACGCTAACGTGAAGTCCGCCTCCGGCGCTAACTAC
Slovenia              ttgcgcgatgttactgggaattttaacgctaatgtcaaatcagccagtggcgcgaactat
                      ** * ** ** ** ** ** ** ******** ** ** ** ***   ***** *****

LifeTech              AACGCTGTGATCGCCTCTGGCAACCAGAGCCTGGGAAGCGGCGTGACCACACTGAGAGCT
Slovenia              aacgccgtgatcgctagcggtaaccaaagcttgggatctggggttacaaccttaagagct
                      ***** ********    ** ***** *** *****   ** ** ** **  * ******

LifeTech              GTGGCCAACGGCAAGACCACCGACCCCCTGAAGGCTCTGGACGACGCTATCGCCTCCGTG
Slovenia              gttgcaaatggtaaaaccacggatccgctgaaagcgctggacgatgctatcgcatctgta
                      ** ** ** ** ** ***** ** ** ***** ** ******** ******** ** **

LifeTech              GACAAGTTCAGATCCAGCCTGGGCGCTGTGCAGAACAGACTGGACAGCGCCGTGACCAAC
Slovenia              gacaaattccgttcttccctcggtgcggtgcaaaaccgtctggattccgcggttaccaac
                      ***** *** * **   *** ** ** ***** *** * *****   *** ** ******

LifeTech              CTGAACAACACCACCACCAACCTGTCCGAGGCCCAGAGCAGAATCCAGGATGCCGACTAC
Slovenia              ctgaacaacaccactaccaacctgtctgaagcgcagtcccgtattcaggacgccgactat
                      ************** *********** ** ** ***  * * ** ***** ********

LifeTech              GCCACCGAGGTGTCCAACATGAGCAAGGCCCAGATCATCCAGCAGGCCGGCAACTCTGTG
Slovenia              gcgaccgaagtgtccaatatgtcgaaagcgcagatcatccagcaggccggtaactccgtg
                      ** ***** ******** ***   ** ** ******************** ***** ***

LifeTech              CTGGCTAAGGCCAACCAGGTGCCCCAGCAGGTGCTGAGTCTGCTGCAGGGA---------
Slovenia              ttggcaaaagctaaccaggtaccgcagcaggttctgtctctgttacagggttactagagc
                      **** ** ** ******** ** ******** ***  **** * *****

LifeTech              -----------------------------
Slovenia              gaggagaccaccaccaccaccaccactag

There are fewer restriction sites in the sequence generated by GeneOptimizer. I don't think that's proof of anything, but seems generally positive. (The images below are from the NEBcutter online tool.)

NEB 1 NEB 2

Comparing Codon Optimizers

I compared three methods of codon optimization: JCAT, IDT and GeneOptimizer. In all three cases I select for Mus musculus as the target organism. There is good concordance between all three, with LifeTech and JCAT at over 90% condordance. Note the addiitonal leader sequence in the LifeTech version — that's the added Kozak sequence.

CLUSTAL O(1.2.1) multiple sequence alignment


IDT           ------ATGGCACAGGTCATTAATACTAATAGTCTGAGTCTGATCACTCAGAATAACATC
LifeTech      GCCACCATGGCCCAAGTGATCAACACCAACAGCCTGAGCCTGATCACCCAGAACAACATC
JCAT          ------ATGGCCCAGGTGATCAACACCAACAGCCTGAGCCTGATCACCCAGAACAACATC
                    *****.**.** ** ** ** ** ** ***** ******** ***** ******

IDT           AACAAAAACCAATCAGCCCTGTCAAGTTCCATAGAGAGGCTGAGTAGCGGACTGAGGATA
LifeTech      AACAAGAACCAGAGCGCCCTGAGCAGCAGCATCGAGAGACTGAGCAGCGGCCTGAGAATC
JCAT          AACAAGAACCAGAGCGCCCTGAGCAGCAGCATCGAGAGGCTGAGCAGCGGCCTGAGGATC
              *****.*****.: .******: .** : ***.*****.***** *****.*****.**.

IDT           AACTCAGCAAAAGATGATGCTGCTGGTCAGGCTATAGCCAACCGGTTCACGTCTAACATA
LifeTech      AACAGCGCCAAGGACGATGCCGCTGGCCAGGCTATCGCCAACAGATTCACCAGCAACATC
JCAT          AACAGCGCCAAGGACGACGCCGCCGGCCAGGCCATCGCCAACAGGTTCACCAGCAACATC
              ***: .**.**.** ** ** ** ** ***** **.******.*.***** :  *****.

IDT           AAAGGATTGACCCAGGCTGCCCGGAACGCTAATGATGGAATCAGCGTGGCCCAGACTACA
LifeTech      AAGGGCCTGACCCAGGCCGCCAGAAACGCCAACGATGGCATCAGCGTGGCCCAGACAACA
JCAT          AAGGGCCTGACCCAGGCCGCCAGGAACGCCAACGACGGCATCAGCGTGGCCCAGACCACC
              **.**. ********** ***.*.***** ** ** **.***************** **.

IDT           GAAGGCGCTTTGAGTGAAATCAACAATAACCTCCAAAGAGTGAGAGAATTGACTGTGCAG
LifeTech      GAGGGCGCACTGAGCGAGATCAACAACAACCTGCAGAGAGTGCGCGAGCTGACCGTGCAG
JCAT          GAGGGCGCCCTGAGCGAGATCAACAACAACCTGCAGAGGGTGAGGGAGCTGACCGTGCAG
              **.*****  **** **.******** ***** **.**.***.* **. **** ******

IDT           GCTACTACTGGAACAAATAGCGAGTCCGATCTGTCTAGCATCCAGGATGAAATTAAATCC
LifeTech      GCCACCACAGGCACAAACAGCGAGAGCGACCTGTCCAGCATCCAGGACGAGATCAAGAGC
JCAT          GCCACCACCGGCACCAACAGCGAGAGCGACCTGAGCAGCATCCAGGACGAGATCAAGAGC
              ** ** ** **.**.** ******: *** ***:  *********** **.** **.: *

IDT           AGACTGGATGAAATCGATCGCGTGAGCGGACAAACTCAGTTTAACGGAGTGAATGTTCTG
LifeTech      AGACTGGATGAGATCGACAGAGTGTCCGGCCAGACCCAGTTCAACGGCGTGAACGTGCTG
JCAT          AGGCTGGACGAGATCGACAGGGTGAGCGGCCAGACCCAGTTCAACGGCGTGAACGTGCTG
              **.***** **.***** .* ***: ***.**.** ***** *****.***** ** ***

IDT           GCTAAAAATGGCAGTATGAAGATCCAGGTCGGAGCCAACGACAACCAGACAATTACAATC
LifeTech      GCCAAGAACGGCAGCATGAAGATCCAAGTGGGCGCCAACGACAACCAGACCATCACCATC
JCAT          GCCAAGAACGGCAGCATGAAGATCCAGGTGGGCGCCAACGACAACCAGACCATCACCATC
              ** **.** ***** ***********.** **.*****************.** **.***

IDT           GACCTCAAGCAAATCGACGCAAAGACGCTGGGCCTGGATGGATTTTCTGTGAAGGCATTG
LifeTech      GACCTGAAGCAGATCGACGCCAAGACCCTGGGCCTGGACGGCTTCTCTGTGAAGGCCCTG
JCAT          GACCTGAAGCAGATCGACGCCAAGACCCTGGGCCTGGACGGCTTCAGCGTGAAGGCCCTG
              ***** *****.********.***** *********** **.** :  ********. **

IDT           ATAACCGCCTCCGGCGATATTTCTTTGACTTTCAAACAGGTCGACGGGGTCAACGACGTC
LifeTech      ATCACAGCCAGCGGCGACATCAGCCTGACCTTCAAACAGGTGGACGGCGTGAACGACGTG
JCAT          ATCACCGCCAGCGGCGACATCAGCCTGACCTTCAAGCAGGTGGACGGCGTGAACGACGTG
              **.**.***: ****** ** :   **** *****.***** ***** ** ********

IDT           ACCTTGGAAAGCGTAAAAGTGTCATCATCAGCAGGCACAGGGATTGGCGTGCTGGCTGAG
LifeTech      ACCCTGGAAAGCGTGAAGGTGTCCAGCTCTGCCGGCACAGGCATCGGCGTGCTGGCTGAA
JCAT          ACCCTGGAGAGCGTGAAGGTGAGCAGCAGCGCCGGCACCGGCATCGGCGTGCTGGCCGAG
              *** ****.*****.**.***: .: .:  **.*****.** ** *********** **.

IDT           GTGATCAACAAGAATTCCAATAGGACCGGAGTGAAGGCCTATGCATCAGTGATAACTACC
LifeTech      GTGATTAACAAGAACAGCAACAGGACCGGCGTGAAGGCCTACGCCAGCGTGATCACCACA
JCAT          GTGATCAACAAGAACAGCAACAGGACCGGCGTGAAGGCCTACGCCAGCGTGATCACCACC
              ***** ******** : *** ********.*********** **.: .*****.** **.

IDT           TCCGATGTGGCTGTCCAGTCCGGGTCCCTGAGCAATCTGACCCTGAACGGCATTCACCTG
LifeTech      AGCGACGTGGCCGTGCAGAGCGGCAGCCTGTCTAACCTGACCCTGAACGGCATCCACCTG
JCAT          AGCGACGTGGCCGTGCAGAGCGGCAGCCTGAGCAACCTGACCCTGAACGGCATCCACCTG
              : *** ***** ** ***: *** : ****:  ** ***************** ******

IDT           GGCAACATCGCAGATATTAAAAAGAACGACTCTGACGGACGCCTTGTGGCAGCAATCAAT
LifeTech      GGCAATATCGCCGATATCAAGAAGAACGACAGCGACGGCAGACTGGTGGCCGCCATCAAC
JCAT          GGCAACATCGCCGACATCAAGAAGAACGACAGCGACGGCAGGCTGGTGGCCGCCATCAAC
              ***** *****.** ** **.*********:  *****..* ** *****.**.*****

IDT           GCTGTCACATCCGAGACTGGAGTGGAAGCCTACACTGATCAGAAAGGCCGACTTAACTTG
LifeTech      GCCGTGACAAGCGAGACAGGCGTGGAAGCCTACACCGACCAGAAGGGCAGACTGAACCTG
JCAT          GCCGTGACCAGCGAGACCGGCGTGGAGGCCTACACCGACCAGAAGGGCAGGCTGAACCTG
              ** ** **.: ****** **.*****.******** ** *****.***.*.** *** **

IDT           AGGTCTATCGACGGAAGAGGTATCGAAATCAAGACCGATAGTGTGTCCAATGGCCCCTCC
LifeTech      AGATCCATCGACGGCAGGGGCATCGAAATCAAGACCGACAGCGTGTCCAACGGCCCCTCC
JCAT          AGGAGCATCGACGGCAGGGGCATCGAGATCAAGACCGACAGCGTGAGCAACGGCCCCAGC
              **.:  ********.**.** *****.*********** ** ***: *** ******: *

IDT           GCACTTACTATGGTGAACGGGGGACAGGATCTCACCAAAGGGAGCACCAACTATGGCAGA
LifeTech      GCCCTGACAATGGTCAACGGCGGACAGGACCTGACCAAGGGCAGCACCAACTACGGCAGG
JCAT          GCCCTGACCATGGTGAACGGCGGCCAGGACCTGACCAAGGGCAGCACCAACTACGGCAGG
              **.** ** ***** ***** **.***** ** *****.** *********** *****.

IDT           CTGTCATTGACTAGACTGGATGCTAAGAGCATCAATGTTGTGTCTGCTTCTGACTCCCAA
LifeTech      CTGTCCCTGACAAGACTGGACGCCAAGTCCATCAACGTGGTGTCCGCCAGCGACTCCCAG
JCAT          CTGAGCCTGACCAGGCTGGACGCCAAGAGCATCAACGTGGTGAGCGCCAGCGACAGCCAG
              ***: . **** **.***** ** ***: ****** ** ***:  ** :  ***: ***.

IDT           CATCTGGGCTTCACCGCCATTGGTTTCGGCGAGTCCCAGGTCGCCGAAACTACTGTAAAT
LifeTech      CACCTGGGCTTTACAGCTATCGGCTTCGGCGAGAGCCAGGTGGCCGAGACTACCGTGAAT
JCAT          CACCTGGGCTTCACCGCCATCGGCTTCGGCGAGAGCCAGGTGGCCGAGACCACCGTGAAC
              ** ******** **.** ** ** *********: ****** *****.** ** **.**

IDT           TTGCGAGATGTGACCGGAAATTTTAATGCCAACGTCAAATCAGCCAGTGGCGCAAACTAC
LifeTech      CTGAGGGACGTGACCGGCAACTTCAACGCTAACGTGAAGTCCGCCTCCGGCGCTAACTAC
JCAT          CTGAGGGACGTGACCGGCAACTTCAACGCCAACGTGAAGAGCGCCAGCGGCGCCAACTAC
              **.*.** ********.** ** ** ** ***** **.: .***:  ***** ******

IDT           AATGCTGTGATCGCTAGTGGAAACCAATCTCTTGGCAGTGGGGTAACTACTCTGAGGGCT
LifeTech      AACGCTGTGATCGCCTCTGGCAACCAGAGCCTGGGAAGCGGCGTGACCACACTGAGAGCT
JCAT          AACGCCGTGATCGCCAGCGGCAACCAGAGCCTGGGCAGCGGCGTGACCACCCTGAGGGCC
              ** ** ******** :  **.*****.:  ** **.** ** **.** ** *****.**

IDT           GTGGCCAACGGCAAGACCACAGATCCCCTGAAGGCCCTCGATGACGCAATAGCCTCTGTG
LifeTech      GTGGCCAACGGCAAGACCACCGACCCCCTGAAGGCTCTGGACGACGCTATCGCCTCCGTG
JCAT          GTGGCCAACGGCAAGACCACCGACCCCCTGAAGGCCCTGGACGACGCCATCGCCAGCGTG
              ********************.** *********** ** ** ***** **.***:  ***

IDT           GATAAATTTAGAAGTAGCCTTGGAGCCGTGCAGAATCGACTCGATAGCGCTGTTACAAAT
LifeTech      GACAAGTTCAGATCCAGCCTGGGCGCTGTGCAGAACAGACTGGACAGCGCCGTGACCAAC
JCAT          GACAAGTTCAGGAGCAGCCTGGGCGCCGTGCAGAACAGGCTGGACAGCGCCGTGACCAAC
              ** **.** **.:  ***** **.** ******** .*.** ** ***** ** **.**

IDT           CTGAATAACACCACCACAAATCTGTCTGAAGCACAGTCACGCATTCAGGATGCTGATTAT
LifeTech      CTGAACAACACCACCACCAACCTGTCCGAGGCCCAGAGCAGAATCCAGGATGCCGACTAC
JCAT          CTGAACAACACCACCACCAACCTGAGCGAGGCCCAGAGCAGGATCCAGGACGCCGACTAC
              ***** ***********.** ***:  **.**.***: ..* ** ***** ** ** **

IDT           GCAACAGAAGTTAGCAATATGTCCAAGGCCCAAATCATCCAACAGGCTGGGAACAGCGTC
LifeTech      GCCACCGAGGTGTCCAACATGAGCAAGGCCCAGATCATCCAGCAGGCCGGCAACTCTGTG
JCAT          GCCACCGAGGTGAGCAACATGAGCAAGGCCCAGATCATCCAGCAGGCCGGCAACAGCGTG
              **.**.**.** : *** ***: *********.********.***** ** ***:  **

IDT           CTTGCCAAAGCTAATCAGGTACCACAGCAGGTATTGTCCCTGCTTCAGGGG
LifeTech      CTGGCTAAGGCCAACCAGGTGCCCCAGCAGGTGCTGAGTCTGCTGCAGGGA
JCAT          CTGGCCAAGGCCAACCAGGTGCCCCAGCAGGTGCTGAGCCTGCTGCAGGGC
              ** ** **.** ** *****.**.********. **:  ***** *****
Comment
Brian Naughton | Wed 13 August 2014 | biotech | DNA vaccine

Aldevron, which seems like an awesome company, is giving away free samples of 2µg of DNA vaccine. I could grow that up to make more of course, but how much DNA do I actually need to inject to elicit a reasonable humoral response? The answer is not straightforward. Wikipedia says the following:

Saline injections require variable amounts of DNA, from 10μg-1 mg, whereas gene gun deliveries require 100 to 1000 times less DNA than intramuscular saline injection to raise an effective immune response. Generally, 0.2μg to 20μg are required, although quantities as low as 16ng have been reported.

One paper on DNA tattooing used 50μg of DNA per injection.

For DNA vaccines, it seems like more DNA is always better. Thankfully, 500μg of DNA is a reasonable amount of DNA to grow up since, for example, this Qiagen Plasmid Maxi Kit or this PureLink HiPure Plasmid Maxiprep Kit each yield 500μg.

Comment

Via Life Technologies website, the pVAX1 vector sequence. It's 2999nt long.

GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT
ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAG
TAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCC
CCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC
ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCA
ATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA
GGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAG
CGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGG
CCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCA
CTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGG
GGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAG
CTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCA
AGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCA
CAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATG
AACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCT
GCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACG
CTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGG
ACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTG
CTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACC
CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCC
TTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGC
ACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTC
AATAATAGCACGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTC
GTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCG
CTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAG
TGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA
GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGA
ACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGG
TCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTT
GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT
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Finding a vector

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Finding a vector

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