TABLE 3

Primers used in this study

PrimerSequence (5′–3′)aCharacteristics
AD01-1GGGGGGGACGTCGGCGCGCTAGAG (AatII)Spcr marker cloning
AD01-2GGGTAGGGATAACAGGGTAATCATGCAAATGTCACTAA (I-SceI)Spcr marker cloning
AD02-1GGGGGGGAGCTCTGTCGATTAATGTCGTAATATC (SacI)YBT-020 slpA promoter cloning
AD02-2GGGGGGTCTAGAAATGTAATACTAGTTCCATACTG (XbaI)YBT-020 slpA promoter cloning
AD02-3GGGGGGTCTAGATAAAAGAGATGGAGGTAACTTTTGCATCAAAAAAACCAGGTAATG (XbaI)I-SceI ORF cloning
AD02-4GGGGGGCTGCAGTTATTTCAGGAAAGTTTCGGAGG (PstI)I-SceI ORF cloning
AD03-1GGGGGGTCTAGATAAAAGAGATGGAGGTAACTTATGCCACAATTTGATATATTATGTAAA (XbaI)FLP ORF cloning
AD03-2GGGGGGCTGCAGTTATATGCGTCTATTTATGTAGGATG (PstI)FLP ORF cloning
AD04-1GGGGGGGAGCTCCCATAATCCAGTTTCCTTTATC (SacI)Left arm for pAD04
AD04-2GGGGGGGGTACCCATACTTACTCACCATCCCA (KpnI)Left arm for pAD04
AD04-3GGGGGGGGTACCTAAATATCCTAAAAAATGGGTATG (KpnI)Right arm for pAD04
AD04-4GGGGGGTCTAGATTACCAAATCGTCGAAAGTTC (XbaI)Right arm for pAD04
AD05-1GGGGGGGAATTCACACGGTGTATATGTGTCAAC (EcoRI)Left arm for pAD05
AD05-2GGGGGGGGTACCCATGTTATATCACCTCCTTGT (KpnI)Left arm for pAD05
AD05-3GGGGGGGGTACCTAATCCCATATGATGGCTTAG (KpnI)Right arm for pAD05
AD05-4GGGGGGGGATCCTACAAGTGCATCCTCTACTTC (BamHI)Right arm for pAD05
AD06-1AACTTTATATGAACATAATCAACG (5′ phosphorylated)pAD06 construction
AD06-2TCACAATTCCACACAACATACpAD06 construction
AD06-3GGGGGGGAATTCTAAAAGAGATGGAGGTAACTTATGACAATGATGGATATGAATTT (EcoRI)crtM-crtN cloning
AD06-4GGGGGGGTCGACTTATACGCCCCGCTCAATATC (SalI)crtM-crtN cloning
AD07-1GGGGGGGAATTCGTAGAACGGTTTCTTGGCAC (EcoRI)Left arm for pAD07
AD07-2GGGGGGGGTACCCATAGGAATATATCCTCCTTAG (KpnI)Left arm for pAD07
AD07-3GGGGGGGGTACCTAAATATATATGGCAAAAAGGTC (KpnI)Right arm for pAD07
AD07-4GGGGGGGGATCCAGTTTCAGATCGATTGTCTAC (BamHI)Right arm for pAD07
AD08-1GGGGGGGAGCTCGACAGCAGAGGCTATCCAG (SacI)Left arm for pAD08
AD08-2GGGGGGGGTACCATTTCAACGAATTTTTCAACAGA (KpnI)Left arm for pAD08
AD08-3GGGGGGGGTACCGGGGGGGGATCCAGAAAAAGAGATTATTTGATATCA (KpnI, BamHI)Right arm for pAD08
AD08-4GCACCGTTTCATACTGTAGTGRight arm for pAD08
AD08-5GGGGGGGGTACCATAACTTCGTATAGCATACATTATACGAAGTTATTTTGTAAAAGCTCCGCAAGAG (KpnI, loxP)spo0A cloning with two loxPs
AD08-6GGGGGGGGATCCATAACTTCGTATAATGTATGCTATACGAAGTTATCAACAAAATAAAAAGACCAACG (BamHI, loxP)spo0A cloning with two loxPs
AD09-1GGGGGGGAGCTCTAATAGGTCTTTGTCTCCTTG (SacI)Left arm for pAD09
AD09-2GGGGGGGGTACCCATGTGTATCCTTCTAGGCT (KpnI)Left arm for pAD09
AD09-3GGGGGGGGTACCTGAGAGTAGAAGTAGCAATGG (KpnI)Right arm for pAD09
AD09-4GGGGGGTCTAGACTTGCATCTAACTTGCATAAG (XbaI)Right arm for pAD09
AD10-1GGGGGGGACGTCAGGCATGGCTGGGCATTC (AatII)Left arm for pAD10 and pAD12
AD10-2GGGGGGGAGCTCGAAACATACTCCTTCTCCGA (SacI)Left arm for pAD10 and pAD12
AD10-3GGGGGGGAGCTCGGGGGGAGATCTGATGAAATAAGCTATCCGATTT (SacI, BglII)Right arm for pAD10 and pAD12
AD10-4GGGGGGTCTAGACCTTCTGGAATCATATCAGTG (XbaI)Right arm for pAD10 and pAD12
AD10-5GGGGGGGAGCTCAATTTGAATATTATGGAAAAAGAC (SacI)BMB171_C0536 promoter cloning
AD10-6GGGGGGGGTACCCATACTACTATCCCTCCTTTTG (KpnI)BMB171_C0536 promoter cloning
AD11-1GGGGGGGACGTCGAGTTAGTGAAGGGTGGTC (AatII)Left arm for pAD11 and pAD13
AD11-2GGGGGGGAGCTCGGGGGGAGATCTGTAAACAGCCTCCTTTAACAT (SacI, BglII)Left arm for pAD11 and pAD13
AD11-3GGGGGGGAGCTCATAAGTAAGGAAGCAGCTTTAG (SacI)Right arm for pAD11 and pAD13
AD11-4GGGGGGTCTAGATTTCTCGTGAGAAAGATATAAC (XbaI)Right arm for pAD11 and pAD13
AD12-1GGGGGGGAGCTCGAAACAATGGTATTACAAACG (SacI)BMB171_C4286 promoter cloning
AD12-2GGGGGGGGTACCCATGTTATATCACCTCCTTGT (KpnI)BMB171_C4286 promoter cloning
TS-TET-FGGGGGGAAGCTTCAGAACGGATTGTTGATG (HindIII)Tetr marker-Ts replicon cloning
TS-TET-RGGGGGGAAGCTTTCGAGCGGAGGCCTG (HindIII)Tetr marker-Ts replicon cloning
CRE-FGGGGGGGGTACCATGTCCAATTTACTGACCGTA (KpnI)Cre recombinase ORF cloning
CRE-RGGGGGGAGATCTCTAATCGCCATCTTCCAGC (BglII)Cre recombinase ORF cloning
C4312-FTGGGATTAACACATGTGGAACGAGConfirmation of crtM-crtN insertion
C4312-RAGCAGCTGCAATAATTAAGAAATCConfirmation of crtM-crtN insertion
  • a Restriction sites are underlined, loxP sites are in italics, and start codons are in boldface. ORF, open reading frame.