Hi,

I currently have a gateway compatible lentiviral vector containing ccdB. I wish to clone in a small oligonucleotide cassette, which contains a DNA barcode in the form of a stretch of random nucleotides. Likely the composition ((SW)5N5)2, where S = strong base, W = weak base, N = any nucleotide, and with the cassette also containg stretches of known DNA sequences bookending the barcode. After cloning, I will perform a pooled LR reaction, thus creating a DNA barcoded library. We also plan on performing ssRNA-seq with this library.

Any literature on DNA barcoding would be greatly appreciated! :)

1. How should I clone in the oligonucleotide cassette? Im considering several options:

a. Gibson assembly (likely option)

b. Golden Gate cloning

c. Zero Background Cloning with XcmI

d. Regular Restriction Enzyme Digest

2. How can I select again vectors which did not receive a barcode?

a. Zero Background Cloning removes a ccdB cassette. However I can't use this. Are there other toxin - antitoxin options available? Ive read about parD.

b. Linearlize the vector via double digest, which would remove a third intervening RE site. Introduce the third RE after gibson assembly/ligation and prior to transformation.

3. I would appreciate suggestions for a good bacterial strain for transformation. I need it to be:

a. Highly competent

b. Electrocompetent

c. ccdB survival

d. Unstable DNA propogation (no recombination, Im transforming with a lentiviral library).

e. Containing another antitoxin if 2a is used?

I was considering One Shot™ ccdB Survival™ 2 T1R Competent Cells OR ElectroMAX™ Stbl4™ Competent Cells

Thank you for the help, and any advice on DNA barcoding would be appreciated.