pJKW0474

Vector backbone

pEarleyGate100

Modifications to backbone

pCLV3-hSpCsn1-CLV3ter fragment was first assembled by Gibson Assembly using the following three PCR fragment:
Fragment 1 CLV3 Promoter 1480bp, At genomic DNA as template
F: actgatttgaaaaatctcag aattccggattatccataataa JKW1005
R: CTTATAGTCCAT ttttagagagaaagtgactgagtg JKW1006

Fragment 2 CAS9, pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene 42230) as template
F: Ttctctctaaaa ATGGACTATAAGGACCACGAC JKW1007
R: Caagagattagg ttaCTTTTTCTTTTTTGCCTGG JKW1008

Fragment 3 CLV3 Terminator 1357bp, At genomic DNA as template
F: AAGAAAAAGtaa cctaatctcttgttgctttaaa JKW1009
R: gccaagcttgcatgcctgca gg aattttttaaaaaaatagttaattatc JKW1010

The Gibson assembly reaction as used as template to amply this assembled fragment using primer pair JKW1105 and JKW1010. This fragment together with EcoR I and Sbf I digested pEARLEYGATE100 are used in a new Gibson assembly to make the final construct.

The SbfI site can be used to clone in guide RNA fragment.

Bacterial Resistance(s)

Kanamycin, 50 μg/mL

Growth Temperature

37℃

Growth Strain(s)

NEB Stable

Copy number

Unknown