User talk:Nkuldell:M13 redesign

M13.1:rough sketch

genetic element	promoter	RBS	coding
	start synthesis with
gII	BBa_M13102 (need 5' UTR?)	BBa_M13502	BBa_M13002' (modified to remove gene 10 promoter)
gX	BBa_M13110 (need 5' UTR?)	BBa_M13510	BBa_M13010' (modified to remove gene 5 promoter)
gV	BBa_M13105 (need 5' UTR?)	BBa_M13505	BBa_M13005
gVII		BBa_M13507	BBa_M13007' (modified to remove overlap with gene 9 dwnstm)
gIX		BBa_M13509	BBa_M15009' (modified to remove overlap with gene 8 dwnstm)
gVIII	BBa_M13108 (need 5' UTR?)	BBa_M13508	BBa_M13008
	Transcriptional terminator (if M13K07 part, then need to modify to remove gene 3 promoter)
gIII	BBa_M13103	BBa_M13503	BBa_M13003' (modified to remove gene 6 promoter, change GTG start?)
gVI	BBa_M13106	BBa_M13506	BBa_M13006' (modified to remove gene 1 promoter)
gI	BBa_M13101	BBa_M13501	BBa_M13001' (modified to remove gene 11 RBS, gene 4 promoter, RBS, start)
gXI		BBa_M13511	BBa_M13011' (modified to remove gene 4 promoter, RBS, start)
gIV	BBa_M13104 (need 5' UTR?)	BBa_M13504	BBa_M13004'
	M13K07 ori/KanR/p15a ori
	end synthesis

Note: modified parts codon varied to remove direct repeats.

Coding sequences/replication sequences

genetic element	bp in M13K07 [[1]]	BBa_	notes on standardization
gene II CDS	8268- 831	BBa_M13002	will need to disable internal RBS that allows gX transcription from within gII coding sequence
gene X CDS	496- 831	BBa_M13010	need to unstuff from inside gII
gene V CDS	843-1106	BBa_M13005	RBS overlaps stop of upstream gII, gX
gene VII CDS	1108-1209	BBa_M13007	stop codon overlaps with start of dwstm gIX
gene IX CDS	1206-1304	BBa_M13009	start codon overlaps with stop of upstm gVII stop codon overlaps with start of dwstm gVIII
gene VIII CDS	1301-1522	BBa_M13008	start codon overlaps with stop of upstm gIX might want different BBa with codon varied ORF might want to include silent cloning sites for N-terminal fusions after first Ala of mature protein
gene III CDS	1579-2853	BBa_M13003	start codon is GTG RBS is within transcriptional terminator for upstream gVIII unique BamHI site in domain 2 may be useful for cloning
gene VI CDS	2856-3194	BBa_M13006
gene I CDS	3196-4242	BBa_M13001	will need to disable internal RBS that leads to gXI transcription weach rho-dependent terminator limits gI txn rare codons limit gI tln
gene XI CDS	3916-4242	BBa_M13011	need to unstuff from inside gI
gene IV CDS	4220-5500	BBa_M13004	start codon and RBS are within gI/gXI coding sequence stop codon is within M13 ori on M13K07
M13 origin of replication (portion)	5488-5830
p15A origin of replication (counterclockwise)	6591-6046
Tn903 kanamycin resistance CDS	7956-7141
M13 origin of replication (portion)	8093-8130

RBS as identified in Gene (1980) 11:129-148

Renumbered bp according to M13K07 genome annotation
16S RNA 3'OHAUUCCUCCACUAG--------
Note: All RBS are directly followed by ATG of coding seq then a codon starting with A, except gene 6, gene 7 and gene I which follow ATG with G (genes 1 and 7) or C (gene 6). Genes 1, 6 and 7 are translated only to low levels in vivo and in vitro.

genetic element	bp in M13K07 [[2]]	BBa_	seq
gene II RBS	8252-8267	BBa_M13502	ATCAACCGGGGTACAT
gene X RBS	480-495	BBa_M13510	ATTTGAGGGGGATTCA
gene V RBS	827-842	BBa_M13505	CATAAGGTAATTCACA
gene VII RBS	1092-1107	BBa_M13507	GTTCCGGCTAAGTAAC
gene IX RBS	1190-1205	BBa_M13509	TCGCTGGGGGTCAAAG
gene VIII RBS	1285-1300	BBa_M13508	TAATGGAAACTTCCTC
gene III RBS	1563-1578	BBa_M13503	TTTGGAGA TTTTCAAC
gene VI RBS	2840-2855	BBa_M13506	ATAAGGAGTCTTAATC
gene I RBS	3180-3195	BBa_M13501	GATTGGGATAAATAAT
gene XI RBS	3900-3915	BBa_M13511	AATTTAGGTCAGAAG RBS not identified in Gene 1980 paper
gene IV RBS	4204-4219	BBa_M13504	AAAAAAGGTAATTCAA

Promoters as identified in Gene (1980) 11:129-148

Renumbered bp according to M13K07 genome annotation
-10 is TATAATpu centered around -8 from mRNA initiation point
-35 is TGTTGACAATT centered around -35 from mRNA initiation point
+2 position is often T
Many of these promoters were identified as DNA fragments that could bind RNAP

genetic element	bp in M13K07 [[3]]	BBa_	seq matches to -35 and -10 concensus in bold positions -30, -20, -10 and zero relative to mRNA start in red differences with fd in purple
gene II promoter	8188-8235	BBa_M13102	TATTAACGTTTACAATTTAAATATTTGCTTATACAATCTTCCTGTTTT
gene X promoter	381-428	BBa_M13110	TCTTTTTGAT GCAATCCGCTTTGCTTCTGA CTATAATAGT CAGGGTAA
gene V promoter	786-835	BBa_M13105	CCAACGTCCTGACTGGTATAATGAGCAGTTCTTAAAATCGCATAAGGTA
gene VII promoter			driven from II, X, V promoters (BBa_M13102,_M13110,_M13105)?
gene IX promoter			driven from II, X, V promoters (BBa_M13102,_M13110,_M13105)?
gene VIII promoter	1155-1201	BBa_M13108	AATCTC CGTTGTACTT TGTTTCGCGC TTGGTATAATCGCTGGGGGT C
gene III promoter	1500-1547	BBa_M13103	AATTCACCTCGAAAGCAAGCTGATAAACCGATACAATTAAAGGCTCCT
gene VI	2716-2764	BBa_M13106	GGTAAACCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCC
gene I promoter	3086-3132	BBa_M13101	CCCGTCTAATGCGCT TCCCTGTTTTTATGTTATTCTCTCTGTAAAGG
gene XI promoter			protein from translation re-initiation event within gene I transcript
gene IV promoter	4055-4103	BBa_M13104	TTGATAAATTCACTATTGACTCTTCTCAGCGTCTTAATCTAAGCTATCG

Terminator sequences

Intergenic region I between genes VI and II, contains ori and PS
Intergenic region II between genes VIII and III, contains transcription terminator