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Table 1 List of primers used in the study

From: Molecular characterization of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations

Primers

Sequence (5′ − 3′)

Direction

Degenerate

CYP450-1 F.

CCN(A/C/G/T)TAY(C/T)GGH(A/C/T)GD(G/A/T)TACTGGAGR(A/G)CAG

Forward

CYP450-1R.

AGTCD(G/A/T)GD(G/A/T)CAW(A/T)D(G/A/T)GCCCATTCD(G/A/T)AG

Reverse

CYP450-2 F.

ATATGGGCY(C/T)GATTATGGV(G/A/C)CCTCA

Forward

CYP450-2R.

AGCATH(A/C/T)AGH(A/C/T)GGAGTTGGAGGY(C/T)TG

Reverse

5′ and 3′ RACE

5′ Adapter*

GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA

 

5′ RACE-OUT*

GCTGATGGCGATGAATGAACACTG

Forward

5′ RACE-IN*

CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG

Forward

5′ CYP98-OUT

TGTTATCAGAATTATTGCAATCTCTGT

Reverse

5′ CYP98-IN

AGCCGTAACCTCATCTTCACGAATGG

Reverse

5′ CYP76-OUT

CGTCTCTGTACCAGCTAAAAACATTTCCAC

Reverse

5′ CYP76-IN

GATCTCATGTTCTGATAACTTGGCTGGTT

Reverse

3′ Adapter*

GCGAGCACAGAATTAATACGACTCACTATAGGT12V(G/A/C)N(A/C/T/G)

 

3′ RACE-OUT*

GCGAGCACAGAATTAATACGACT

Reverse

3′ RACE- IN*

CGCGGATCCGAATTAATACGACTCACTATAGG

Reverse

3′ CYP98-OUT

TGATCGGCTATGAGCGCGTGATGAA

Forward

3′ CYP98-IN

AAGCGCTAAGGTTGCAGCCTCCAA

Forward

3′ CYP76-OUT

AGAGATCCTGAATGTTGGGATGACCCTAT

Forward

3′ CYP76-IN

AGAGATCCTGAATGTTGGGATGACCCTAT

Forward

Full length

  

FullCYP98F

ATGGCAATTCCCTTAGCTGCTGCAATCCCTC

Forward

FullCYP98R

TTATATGTCCACTGCAATTCGTTTATACAACTCAGC

Reverse

FullCYP76F

ATGGAATGGGAATGGAGCTATTTGT

Forward

FullCYP76R

CTAGATAAGATTGATGAGTGTCTCA

Reverse

Expression ( E. coli )

CYP98BamHIF

CGGGATCCATGGCAATTCCCTTAGCTGCTGCAA

Forward

CYP98SalIR

GCTCGTCGACTTATATGTCCACTGCAATTCGT

Reverse

CYP76BamHIF

CGGGATCCATGGAATGGGAATGGAGCTATTTGT

Forward

CYP76SalIR

GCTCGTCGACCTAGATAAGATTGATGAGTGTCTCA

Reverse

Real-Time analysis

RTCYP98F

GTGATGGATGAACAAGGAAACGA

Forward

RTCYP98R

CCGTGCTTAGAAAATGCATCCTC

Reverse

RTCYP76 F.P

TAGAATGGGCACTAGCAGAGCTTTTGCG

Forward

RTCYP76 R.P

CTTGCATATAAGGGAGATTGTCGATGTCGTT

Reverse

ActinF

GAGAGTTTTGATGTCCCTGCCATG

Forward

ActinR

CAACGTCGCATTTCATGATGGAGT

Reverse

  1. *Primers marked with star were provided with the kits. Added restriction sites for cloning into pGEX4T-2 vector are bold.
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BMC Biotechnology

ISSN: 1472-6750

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