Table 2 Primers and oligos used in this study
From: An asymmetric PCR-based, reliable and rapid single-tube native DNA engineering strategy
Primer | Sequence(5′-3′) | Comments |
|---|---|---|
KanR-F | agctatgaccatgattacgGGCccTAGCGGTCACGCTGCGCGTAACC | Underlined is the artificial ApaI (GGGCCC) site; the two primers were for amplifying 1.6 kb kanR cassette from pIRES2-EGFP |
KanR-R | gtcgacctgcaggcatgcaagctt CAAACGACCCAACACCGTGCG | |
KanR-Right-15 | atcatggtcatagct | incremental homolog length from 15 bp to 45 bp |
KanR-Right-20 | ccgtaatcatggtcatagct | |
KanR-Right-25 | agggcccgtaatcatggtcatagct | |
KanR-Right-30 | ccgcta gggcccgtaatcatggtcatagct | |
KanR-Right-35 | cgtga ccgcta gggcccgtaatcatggtcatagct | |
KanR-Right-40 | cgcag cgtga ccgcta gggcccgtaatcatggtcatagct | |
KanR-Right-45 | ttacg cgcag cgtga ccgcta gggcccgtaatcatggtcatagct | |
KanR-F-2 kb | agctatgaccatgattacgggccc AACCAATAGGCCGAAATCGGC | incremental insert length |
KanR-F-2.5 kb | agctatgaccatgattacgggccc TCGCCGACCACTACCAGCAG | |
KanR-F-3 kb | agctatgaccatgattacgggccc ACCGGGGTGGTGCCCATCC | |
KanR-F-4 kb | agctatgaccatgattacgggccc GGCATTATGCCCAGTACATGACC | |
pF(3.8 kb) | gctcgagatc tgcgatctaa gtaagcttgg CATCATTAAACTTCTGACAAGCC | 3.8 kb porcine MSTN promoter region |
pF(2.3 kb) | gctcgagatc tgcgatctaa gtaagcttgg GTGCCATGAGTATTGATTCTGGAG | 2.3 kb porcine MSTN promoter region |
pR(promoter) | catggtggctttaccaacagtaccggaatg CGCCAAGCAAAATTTTAATGCC | porcine MSTN promoter region |
P1R(promoter) | ttagatcgcagatctcgagc | 3′ outermost primer for promoter |
pF(1.4 kb) | gcagacatga taagatacat tgatg GGTTCATTACTTCCTAAAACATGG | porcine MSTN terminator |
pR(terminator) | CTCTCAAGGG CATCGGTCGA CGGATCC GTTTCTACACATTAGATGTAAG | |
P1R(terminator) | CATCA ATGTATCTTA TCATGTCTGC | 3′ outermost primer for terminator |
MSTN-P-F | gaattcgagctcggtacccgg CATCATTAAACTTCTGACAAGCC | construction of porcine MSTN expression cassette driving the expression of EGFP |
MSTN-P-R | gcaggtcgactctagaggatcc GCCAAGCAAAATTTTAATGCC | |
MSTN-P-Right | ccgggtaccgagctcgaattc | |
MSTN-T-F | aagcttggcgtaatcatggtc ATTTATATTTGGTTCATTACTTCC | |
MSTN-T-R | tttcacacaggaaacagctat CTTACATCTAATGTGTAGAAAC | |
MSTN-T-Right | gaccatgattacgccaagctt | |
EGFP-F | CATCATTAAACTTCTGACAAGCC ACCATGGTGAGCAAGGGCG | |
EGFP-R | GGAAGTAATGAACCAAATATAAAT TTACTTGTACAGCTCGTCCATGCC | |
EGFP-Right | GGCTTGTCAGAAGTTTAATGATG |