Construction of Zebrafish 7.4 kb lck promoter Gateway 5’ entry clone.

Using BAC clone CH211-135B6, we PCR amplified 5.5 kb of the promoter immediately adjacent to the zebrafish lymphocyte-specific protein tyrosine kinase (lck) start codon and cloned this into pDONR™P4-P1R (Invitrogen) via Gateway® BP Clonase® II enzyme mix to generate the 5.5kb lck promoter entry clone. At the 5' end of this 5.5 kb segment we modified a single nucleotide (T->C) to create a novel SfiI restriction site.

PCR primers are listed below. Lower case letters indicate lck target sequence. Upper case letters indicate overhang sequences.

>lck_prom_Forward_attB4
GGGGACAACTTTGTATAGAAAAGTTGggcccctgaggcccccaagacacccatttttg

>lck_prom_Reverse_attB1
GGGGACTGCTTTTTTGTACAAACTTGgtttactaagcatgagagaaaatggtgcaac

We then PCR amplified an additional 1.9 kb of the lck promoter and ligated it into the SfiI restriction site.

PCR primers are listed below. Lower case letters indicate lck target sequence. Upper case letters indicate overhang sequences.

>lck_prom_Forward_SfiI
GCATGCGGCCACTGAGGCCtgcgcaacagttctagtgctattgaagtgg

>lck_prom_Reverse_SfiI
CTTGGGGGCCTCAGGGGCCacaagggggtgctgggagaaaatttgaaaag