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 |
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