Help:Standards/Assembly/Type IIS/Fusion Sites

This page is still in draft. New information is currently being added to this page.

A Type IIS assembly standard, like MoClo, specifies the overhangs created by a Type IIS enzyme as fusion sites. Fusion sites flank part samples based on position/type, and when assembled, fall in between parts. This standardization allows for reuse and sharing of part samples. There are several Type IIS assembly standards each with their own set of fusion sites.

However, one strength of Type IIS assembly is that users can define their own overhangs as well, creating internal standards or ones specific to a single assembly. This allows for more flexibility in assembly, both in quantity of parts assembled and creating "scarless" junctions. This does come at the cost of making these part samples less "shareable" as another user may not be able to use them in the same context.

Type IIS enzymes like Bsa1, leave 4bp overhangs. This means that there are 246 (4⁴) possible overhangs/fusion sites. The following is a table of all possible overhangs. There are a few recommendations to choosing overhangs for an assembly, including avoiding palindromic ones, to prevent an overhang ligating to itself in an unintended orientation.

ID	Overhang	Complement	Palindrome
1	AAAA	TTTT	no
2	AAAC	TTTG	no
3	AAAG	TTTC	no
4	AAAT	TTTA	no
5	AACA	TTGT	no
6	AACC	TTGG	no
7	AACG	TTGC	no
8	AACT	TTGA	no
9	AAGA	TTCT	no
10	AAGC	TTCG	no
11	AAGG	TTCC	no
12	AAGT	TTCA	no
13	AATA	TTAT	no
14	AATC	TTAG	no
15	AATG	TTAC	no
16	AATT	TTAA	yes
17	ACAA	TGTT	no
18	ACAC	TGTG	no
19	ACAG	TGTC	no
20	ACAT	TGTA	no
21	ACCA	TGGT	no
22	ACCC	TGGG	no
23	ACCG	TGGC	no
24	ACCT	TGGA	no
25	ACGA	TGCT	no
26	ACGC	TGCG	no
27	ACGG	TGCC	no
28	ACGT	TGCA	yes
29	ACTA	TGAT	no
30	ACTC	TGAG	no
31	ACTG	TGAC	no
32	ACTT	TGAA	no
33	AGAA	TCTT	no
34	AGAC	TCTG	no
35	AGAG	TCTC	no
36	AGAT	TCTA	no
37	AGCA	TCGT	no
38	AGCC	TCGG	no
39	AGCG	TCGC	no
40	AGCT	TCGA	yes
41	AGGA	TCCT	no
42	AGGC	TCCG	no
43	AGGG	TCCC	no
44	AGGT	TCCA	no
45	AGTA	TCAT	no
46	AGTC	TCAG	no
47	AGTG	TCAC	no
48	AGTT	TCAA	no
49	ATAA	TATT	no
50	ATAC	TATG	no
51	ATAG	TATC	no
52	ATAT	TATA	yes
53	ATCA	TAGT	no
54	ATCC	TAGG	no
55	ATCG	TAGC	no
56	ATCT	TAGA	no
57	ATGA	TACT	no
58	ATGC	TACG	no
59	ATGG	TACC	no
60	ATGT	TACA	no
61	ATTA	TAAT	no
62	ATTC	TAAG	no
63	ATTG	TAAC	no
64	ATTT	TAAA	no
65	CAAA	GTTT	no
66	CAAC	GTTG	no
67	CAAG	GTTC	no
68	CAAT	GTTA	no
69	CACA	GTGT	no
70	CACC	GTGG	no
71	CACG	GTGC	no
72	CACT	GTGA	no
73	CAGA	GTCT	no
74	CAGC	GTCG	no
75	CAGG	GTCC	no
76	CAGT	GTCA	no
77	CATA	GTAT	no
78	CATC	GTAG	no
79	CATG	GTAC	yes
80	CATT	GTAA	no
81	CCAA	GGTT	no
82	CCAC	GGTG	no
83	CCAG	GGTC	no
84	CCAT	GGTA	no
85	CCCA	GGGT	no
86	CCCC	GGGG	no
87	CCCG	GGGC	no
88	CCCT	GGGA	no
89	CCGA	GGCT	no
90	CCGC	GGCG	no
91	CCGG	GGCC	yes
92	CCGT	GGCA	no
93	CCTA	GGAT	no
94	CCTC	GGAG	no
95	CCTG	GGAC	no
96	CCTT	GGAA	no
97	CGAA	GCTT	no
98	CGAC	GCTG	no
99	CGAG	GCTC	no
100	CGAT	GCTA	no
101	CGCA	GCGT	no
102	CGCC	GCGG	no
103	CGCG	GCGC	yes
104	CGCT	GCGA	no
105	CGGA	GCCT	no
106	CGGC	GCCG	no
107	CGGG	GCCC	no
108	CGGT	GCCA	no
109	CGTA	GCAT	no
110	CGTC	GCAG	no
111	CGTG	GCAC	no
112	CGTT	GCAA	no
113	CTAA	GATT	no
114	CTAC	GATG	no
115	CTAG	GATC	yes
116	CTAT	GATA	no
117	CTCA	GAGT	no
118	CTCC	GAGG	no
119	CTCG	GAGC	no
120	CTCT	GAGA	no
121	CTGA	GACT	no
122	CTGC	GACG	no
123	CTGG	GACC	no
124	CTGT	GACA	no
125	CTTA	GAAT	no
126	CTTC	GAAG	no
127	CTTG	GAAC	no
128	CTTT	GAAA	no
129	GAAA	CTTT	no
130	GAAC	CTTG	no
131	GAAG	CTTC	no
132	GAAT	CTTA	no
133	GACA	CTGT	no
134	GACC	CTGG	no
135	GACG	CTGC	no
136	GACT	CTGA	no
137	GAGA	CTCT	no
138	GAGC	CTCG	no
139	GAGG	CTCC	no
140	GAGT	CTCA	no
141	GATA	CTAT	no
142	GATC	CTAG	yes
143	GATG	CTAC	no
144	GATT	CTAA	no
145	GCAA	CGTT	no
146	GCAC	CGTG	no
147	GCAG	CGTC	no
148	GCAT	CGTA	no
149	GCCA	CGGT	no
150	GCCC	CGGG	no
151	GCCG	CGGC	no
152	GCCT	CGGA	no
153	GCGA	CGCT	no
154	GCGC	CGCG	yes
155	GCGG	CGCC	no
156	GCGT	CGCA	no
157	GCTA	CGAT	no
158	GCTC	CGAG	no
159	GCTG	CGAC	no
160	GCTT	CGAA	no
161	GGAA	CCTT	no
162	GGAC	CCTG	no
163	GGAG	CCTC	no
164	GGAT	CCTA	no
165	GGCA	CCGT	no
166	GGCC	CCGG	yes
167	GGCG	CCGC	no
168	GGCT	CCGA	no
169	GGGA	CCCT	no
170	GGGC	CCCG	no
171	GGGG	CCCC	no
172	GGGT	CCCA	no
173	GGTA	CCAT	no
174	GGTC	CCAG	no
175	GGTG	CCAC	no
176	GGTT	CCAA	no
177	GTAA	CATT	no
178	GTAC	CATG	yes
179	GTAG	CATC	no
180	GTAT	CATA	no
181	GTCA	CAGT	no
182	GTCC	CAGG	no
183	GTCG	CAGC	no
184	GTCT	CAGA	no
185	GTGA	CACT	no
186	GTGC	CACG	no
187	GTGG	CACC	no
188	GTGT	CACA	no
189	GTTA	CAAT	no
190	GTTC	CAAG	no
191	GTTG	CAAC	no
192	GTTT	CAAA	no
193	TAAA	ATTT	no
194	TAAC	ATTG	no
195	TAAG	ATTC	no
196	TAAT	ATTA	no
197	TACA	ATGT	no
198	TACC	ATGG	no
199	TACG	ATGC	no
200	TACT	ATGA	no
201	TAGA	ATCT	no
202	TAGC	ATCG	no
203	TAGG	ATCC	no
204	TAGT	ATCA	no
205	TATA	ATAT	yes
206	TATC	ATAG	no
207	TATG	ATAC	no
208	TATT	ATAA	no
209	TCAA	AGTT	no
210	TCAC	AGTG	no
211	TCAG	AGTC	no
212	TCAT	AGTA	no
213	TCCA	AGGT	no
214	TCCC	AGGG	no
215	TCCG	AGGC	no
216	TCCT	AGGA	no
217	TCGA	AGCT	yes
218	TCGC	AGCG	no
219	TCGG	AGCC	no
220	TCGT	AGCA	no
221	TCTA	AGAT	no
222	TCTC	AGAG	no
223	TCTG	AGAC	no
224	TCTT	AGAA	no
225	TGAA	ACTT	no
226	TGAC	ACTG	no
227	TGAG	ACTC	no
228	TGAT	ACTA	no
229	TGCA	ACGT	yes
230	TGCC	ACGG	no
231	TGCG	ACGC	no
232	TGCT	ACGA	no
233	TGGA	ACCT	no
234	TGGC	ACCG	no
235	TGGG	ACCC	no
236	TGGT	ACCA	no
237	TGTA	ACAT	no
238	TGTC	ACAG	no
239	TGTG	ACAC	no
240	TGTT	ACAA	no
241	TTAA	AATT	yes
242	TTAC	AATG	no
243	TTAG	AATC	no
244	TTAT	AATA	no
245	TTCA	AAGT	no
246	TTCC	AAGG	no
247	TTCG	AAGC	no
248	TTCT	AAGA	no
249	TTGA	AACT	no
250	TTGC	AACG	no
251	TTGG	AACC	no
252	TTGT	AACA	no
253	TTTA	AAAT	no
254	TTTC	AAAG	no
255	TTTG	AAAC	no
256	TTTT	AAAA	no