48 base attP site flanked by 70 bases of target DNA. Dilute in
water or 10 mM Tris–HCl, pH 8.0 to a final concentration of
300 ng/μL.
3. Orbit integration plasmids: attB-containing vectors to be coelectroporated with attP-containing oligonucleotides for construction of deletions and fusions. For a complete list of attBcontaining integration plasmids, see Table 1. They are available
at Addgene.com. Purify these plasmids using a Qiagen PCR
Purification kit (or similar kit) and include an additional washing step to the manufacturer’s instructions (see Note 2). Elute
in sterile water or 10 mM Tris–HCl, pH 8.0.
Table 1
ORBIT integration plasmids
Plasmid
name
Type of modification
Drug resistance
markers
Addgene
ID
C-terminal tags:
pKM446
C-terminal tag: Flag-DAS tag
Hyg
R
108321
pKM468
C-terminal tag: EGFP-4xGly-TEV-Flag-6xHis
Hyg
R
108434
pKM469
C-terminal tag: Venus-4xGly-TEV-Flag-6xHis
Hyg
R
108435
pKM489
C terminal tag: SNAP tag
Hyg
R
108437
pKM490
C-terminal tag: CLIP tag
Hyg
R
109281
pKM491
C-terminal tag: 4xGly-TEV-Flag-6xHis
Hyg
R
109282
pKM492
C-terminal tag: 4xGly-TEV-Myc-6xHis
Hyg
R
109283
pKM493
C-terminal tag: TEV-Flag-4xGly-EGFP
Hyg
R
pKM495
C-terminal tag: Flag-DAS tag
Zeo
R
109284
Knockouts:
pKM464
Knockout
Hyg
R
108322
pKM496
Knockout
Zeo
R
109301
Promoter replacements:
pKM464
Replace endogenous promoter with P Hyg
Hyg
R
108322
pKM496
Replace endogenous promoter with P GroEL
(op-rbs)
Zeo
R
109301
pKM508
Replace endogenous promoter with P21 (op-rbs) Zeo
R
pKM509
Replace endogenous promoter with P38 (op-rbs) Zeo
R
Markerless deletions:
pKM512
To excise integrating plasmids, leaving attP site
intact
Zeo
R
Oligo-Mediated Recombineering in Mycobacteria
307