After extensive research, we have found that information about circRNA is limited. Currently, there is not only a lack of effective methods for detecting circRNA, but also no standard protocol for synthesizing it. Therefore, we are providing a coherent platform for both the synthesis and detection of circRNA. Our platform includes following three sections:

1. CircRNA synthesis. (Figure 1)

2. Gold nanoparticle-based colorimetric assay. (Figure 2)

3. Lateral flow test. (Figure 3)

Figure 1. CircRNA synthesis process. We utilize restriction enzymes for traditional cloning (BBa_K4636040, BBa_K4636041), PCR primers (BBa_K4636001 - BBa_K4636006) for amplifying the target (BBa_K4636040, BBa_K4636041), a modified T7 promoter (BBa_K4636000) for efficient transcription of DNA to RNA. Finally, we use a splint (BBa_K4636013 - BBa_K4636018) to assist in the circularization procedure.

Figure 2. Process of gold nanoparticle-based colorimetric assay. We use RCA primers (BBa_K4636028 - BBa_K4636035) to conduct the RCA and generate long-repeated ssDNA for further detection using DNA probes (BBa_K4636007 - BBa_K4636012) on gold nanoparticles.

Figure 3. Process of lateral flow test. We utilize these modified RPA primers (BBa_K4636019 - BBa_K4636027, BBa_K4636042) to conduct RT-RPA, and the resulting amplicon can be detected using PCRD detection.

Item	Number	Name	Description
1	BBa_K4636000	Modified T7 promoter	The modified T7 promoter can be used to transcribe efficiency.
2	BBa_K4636001	cDNA of 0101802	cDNA of hsa_circ_0101802
3	BBa_K4636002	cDNA of 0004771	cDNA of hsa_circ_0004771
4	BBa_K4636003	PCR_0101802fw	Insert_0004771 PCR forward primer
5	BBa_K4636004	PCR_0101802rv	Insert_0004771 PCR reverse primer
6	BBa_K4636005	PCR_0004771fw	Insert_0101802 PCR forward primer
7	BBa_K4636006	PCR_0004771rv	Insert_0101802 PCR reverse primer
8	BBa_K4636007	Probe_0101802_bsj	Probe recognizes back splicing junction of RCA product
9	BBa_K4636008	Probe_0101802_10	Probe for recognizing RCA product
10	BBa_K4636009	Probe_0101802_11	Probe for recognizing RCA product
11	BBa_K4636010	Probe_0004771_bsj	Probe recognizes back splicing junction of RCA product
12	BBa_K4636011	Probe_0004771_11	Probe for recognizing RCA product
13	BBa_K4636012	Probe_0004771_12	Probe for recognizing RCA product
14	BBa_K4636013	Splint_1802_18	Splint helps the back splicing of linear RNA
15	BBa_K4636014	Splint_1802_16	Splint helps the back splicing of linear RNA
16	BBa_K4636015	Splint_1802_14	Splint helps the back splicing of linear RNA
17	BBa_K4636016	Splint_4771_18	Splint helps the back splicing of linear RNA
18	BBa_K4636017	Splint_4771_16	Splint helps the back splicing of linear RNA
19	BBa_K4636018	Splint_4771_14	Splint helps the back splicing of linear RNA
20	BBa_K4636019	RPA_0101802fw_1	With biotin modification, it can be used for lateral flow testing after RPA.
21	BBa_K4636020	RPA_0101802rv_1	With FAM modification, it can be used for lateral flow testing after RPA.
22	BBa_K4636021	RPA_0101802fw_2	With biotin modification, it can be used for lateral flow testing after RPA.
23	BBa_K4636022	RPA_0101802rv_2	With FAM modification, it can be used for lateral flow testing after RPA.
24	BBa_K4636023	RPA_0004771fw_1	With biotin modification, it can be used for lateral flow testing after RPA.
25	BBa_K4636024	RPA_0004771rv_1	With DIG modification, it can be used for lateral flow testing after RPA.
26	BBa_K4636025	RPA_0004771fw_2	With biotin modification, it can be used for lateral flow testing after RPA.
27	BBa_K4636026	RPA_0004771rv_2	With DIG modification, it can be used for lateral flow testing after RPA.
28	BBa_K4636027	RPA_0004771fw_3	With biotin modification, it can be used for lateral flow testing after RPA.
29	BBa_K4636042	RPA_0004771rv_3	With DIG modification, it can be used for lateral flow testing after RPA.
30	BBa_K4636028	RCA_indu_1802_1	RCA primers initiates the amplification process
31	BBa_K4636029	RCA_indu_1802_2	RCA primers initiates the amplification process
32	BBa_K4636030	RCA_indu_4771_1	RCA primers initiates the amplification process
33	BBa_K4636031	RCA_indu_4771_2	RCA primers initiates the amplification process
34	BBa_K4636032	RCA_45_1802_1	RCA primers initiates the amplification process
35	BBa_K4636033	RCA_45_1802_2	RCA primers initiates the amplification process
36	BBa_K4636034	RCA_45_4771_1	RCA primers initiates the amplification process
37	BBa_K4636035	RCA_45_4771_2	RCA primers initiates the amplification process
38	BBa_K4636036	PCRforAELA_fw	Primer for AELA-PCR preparation
39	BBa_K4636037	PCRforAELA_rv	Primer for AELA-PCR preparation
40	BBa_K4636038	AELA_PCR_fw	Primer for AELA-PCR
41	BBa_K4636039	AELA_PCR_rv	Primer for AELA-PCR

Item	Number	Name	Composite
1	BBa_K4636040	Insert_0101802	Modified T7 promoter + cDNA of 0101802
2	BBa_K4636041	Insert_0004771	Modified T7 promoter + cDNA of 0004771
3	BBa_K4636052	insert_ligation_0101802	Insert_0101802 + Insert_0101802
4	BBa_K4636053	insert_ligation_0004771	Insert_0004771 + Insert_0004771
5	BBa_K4636054	pUC19_insert_ligation_0101802	puc19 vector + insert_ligation_0101802
6	BBa_K4636055	pUC19_insert_ligation_0004771	puc19 vector + insert_ligation_0004771