1198 pCDNA3.2-ssRFP-Fc-(3c)

pCDNA3.2-ssRFP-Fc-(3c) (1198)

Date: 2012-05-26

Notebook:  4480

 Page: 4482

Clone ID: 1198

Experimenters: Paul

Cloning

PCR using these primers

Sal1 BamH1 Kozak
RFP-Fc-Sal1-F1 accgtcGTCGACGGATCCGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACC
RFP-Fc-Sal1-F2 CTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGTGTCTAAGGGCGAAGAGCTG
ssRFP accgtcGTCGACGGATCCGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGTGTCTAAGGGCGAAGAGCTG
RFP linker Fc
RFP-Fc fusion CTAGCAAACTGGGGCACAAACT GGC AGC GGC GACAAAACTCACACATGCCCAC
RFP-Fc-F3 CTAGCAAACTGGGGCACAAACT GGC AGC GGC GACAAAACTCACACATGCCCAC
RFP-Fc-R1 GTGGGCATGTGTGAGTTTTGTCGCCGCTGCCAGTTTGTGCCCCAGTTTGCTAG

Fc TEV Kpn1
ctctccctgtctccgggtaaa GGC AGC GGC GAG AAC CTG TAC TTC CAG GGC ggt acc
G S G E N L Y F Q G
Fc-TEV-Kpn1-R4
(ctctccctgtctccgggtaaa GGC AGC GGC GAG AAC CTG TAC TTC CAG GGC ggt acc ctagta)
tactagggtaccGCCCTGGAAGTACAGGTTCTCGCCGCTGCCtttacccggagacagggagag

Fc-HRV3C-Kpn1-R5
Fc human rhinovirus (HRV) type 14 3C protease
(ctctccctgtctccgggtaaa ctg gag gtg ctg ttc cag ! ggc ccc ggt acc ctagta)
L E V L F Q G P G T

tactagggtaccggggccctggaacagcacctccagtttacccggagacagggagag

How to make
1. PCR using RFP-Fc-Sal1-F1, 10% RFP-Fc-Sal1-F2 + RFP-Fc-R1 using 114 pET28b-TagRFP as template --> to amplify RFP
2. PCR using RFP-Fc-F3 + Fc-HRV3C-Kpn1-R5 and 380 pCMVK-IL2-intFc-IRES-TagGFP 1101 pFUSE-hIgG1-Fc2-IAB as template, to amplify Fc domain.
3. Join two fragment using Gibson mix, gel purify
4. Digest with BamH1 + Kpn1.
5. Ligate to BamH1 + Kpn1 digested 1192 pCDNA3.2.

	1	2	3	4	5	6	7	8	9	10
	1199 Gibson	1199 PCR	1199 PCR	1199 Gibson	1199 Gibson	1198 Gibson	1198 Gibson	1198 Gibson	1198 PCR	1198 PCR

5/27/2012

digest PCR product and vector with BamH1 + Kpn1

1	2	3	4
1192 pCDNA3.2	1198 ssRFP-Fc-(3c)	1193 Fc	1199 ssRFP-Fc-(tev)

gel purified, and ligate, transform DH5alpha.

Colony Screening by Restriction Digestion

5-29-2012

clone 5 may be right, need to sequence