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BjornFJohansson / pydna

Clone with Python! Data structures for double stranded DNA & simulation of homologous recombination, Gibson assembly, cut & paste cloning.
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Implement Gateway Cloning #313

Open dgruano opened 1 month ago

dgruano commented 1 month ago

I saw there was a placeholder file for Gateway cloning and thought to give it a go. Did you have any idea on how to do it @BjornFJohansson?

manulera commented 1 month ago

I think we should wait for the new assembly code to be merged into pydna, since ideally future implementations should be based on it, I think.

BjornFJohansson commented 1 month ago

I gathered some information on Gateway cloning that are in the placeholder file. Gateway is to similar to homologous recombination and in some way simpler. I gathered some of the information from looking at the ApE source code.

Some links:

snapgene atts Protocols embl

Some notes I made awhile back:

Function: GatewayDesigner(seqs)

Argument:

seqs: A list of linear Amplicons or linear Dseqrecords. The list should include att site sequences.

Output:

A list of linear Amplicons with primers engineered to permit Gateway assembly

Suggested flow:

Verify the absence of gateway sites in input. Sequences are not expected to have these. Check for:

att1_shared TTTGTACAAAAAAG att2_shared CTTTCTTGTACAAAGT att3_shared CAACTTTATTATACA att4_shared CAACTTTGTATAGAAAAGTTG

Verify the rest of att sequences on each side and identify B, P L and R sites. Raise error or throw exception if present.

Add primer tails for the correct pattern of att sites based on the sequence order.

For example:

... seq1 attB1 seq2 ...

==>

Function: GatewayAssembler(seqs)

Argument:

seqs: A list of linear Amplicons and linear or circular Dseqrecords

Output:

Assembled sequences, linear or circular

Split sequences along atts

Add them to Graph

Find circular and linear paths

...

Specificity:

attP1 + attB1 <=> attR1 + attL1 attP2 + attB2 <=> attR2 + attL2

Sequences

  AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACACATTGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA

AttP1 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTACAAAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA

list of left, overlapping and right side of the recombination site, then the left and right sides of the [[site]] that it recombines with (ex. these will be the left and right sides of the attP1 [[site]] for an attB1 site)

attP1 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMAS TTTGTACAAAAAAG YWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG CMASTWT AAAGYWG attP2 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMAS TTTGTACAAGAAAG YWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG CMASTWT AAAGYWG attP3 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMAS TTTGTATAATAAAG YWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG CMASTWT AAAGYWG attP4 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMAS TTTGTATAGAAAAG YWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG CMASTWT AAAGYWG attP5 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMAS TTTGTATACAAAAG YWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG CMASTWT AAAGYWG

                                                                       attB1 CMASTWT GTACAAA AAAGYWG AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG
                                                                       attB2 CMASTWT GTACAAG AAAGYWG AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG
                                                                       attB3 CMASTWT GTATAAT AAAGYWG AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG
                                                                       attB4 CMASTWT GTATAGA AAAGYWG AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG
                                                                       attB5 CMASTWT GTATACA AAAGYWG AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG

attL1 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT GTACAAA AAAGYWG CMASTWT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG attL2 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT GTACAAG AAAGYWG CMASTWT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG attL3 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT GTATAAT AAAGYWG CMASTWT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG attL4 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT GTATAGA AAAGYWG CMASTWT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG attL5 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT GTATACA AAAGYWG CMASTWT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG

                                                                       attR1 CMASTWT GTACAAA AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT AAAGYWG
                                                                       attR2 CMASTWT GTACAAG AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT AAAGYWG
                                                                       attR3 CMASTWT GTATAAT AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT AAAGYWG
                                                                       attR4 CMASTWT GTATAGA AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT AAAGYWG
                                                                       attR5 CMASTWT GTATACA AAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTG AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTT AAAGYWG

attB5 CMASTWTGTATACAAAAGYWG misc_recomb # dda0dd # dda0dd 0 0
attB4 CMASTWTGTATAGAAAAGYWG misc_recomb # dda0dd # dda0dd 0 0
attB3 CMASTWTGTATAATAAAGYWG misc_recomb # dda0dd # dda0dd 0 0
attB2 CMASTWTGTACAAGAAAGYWG misc_recomb # dda0dd # dda0dd 0 0
attB1 CMASTWTGTACAAAAAAGYWG misc_recomb # dda0dd # dda0dd 0 0
attP5 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTTGTATACAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA misc_recomb # dda0dd # dda0dd 0 0
attP4 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTATAGAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA misc_recomb # dda0dd # dda0dd 0 0
attP3 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTATAATAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA misc_recomb # dda0dd # dda0dd 0 0
AttP2 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTACAAGAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA misc_recomb plum plum 0 0
AttP1 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTACAAAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA misc_recomb plum plum 0 0
attL5 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATKMTTTYTTATAATGCCMASTTTGTATACAAAAGYWG misc_recomb # dda0dd # dda0dd 0 0
attL4 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTATAGAAAAGYWG misc_recomb # dda0dd # dda0dd 0 0
attL3 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTATAATAAAGYWG misc_recomb # dda0dd # dda0dd 0 0
AttL2 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTACAAGAAAGYWG misc_recomb plum plum 0 0
AttL1 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTACAAAAAAGYWG misc_recomb plum plum 0 0
attR5 CMASTWTGTATACAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA misc_recomb # dda0dd # dda0dd 0 0
attR4 CMASTTTGTATAGAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA misc_recomb # dda0dd # dda0dd 0 0
attR3 CMASTTTGTATAATAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA misc_recomb # dda0dd # dda0dd 0 0
AttR2 CMASTTTGTACAAGAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA misc_recomb plum plum 0 0
AttR1 CMASTTTGTACAAAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATRCTGTAAAACACAACATATSCAGTCAYWWTGAATCAACTACTTAGATGGTATTAGTGACCTGTA misc_recomb plum plum 0 0
ccdB ATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCNCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATA CDS yellow yellow 0 0
Gateway Recomb5 cmastwtGTATACAaaagywg misc_feature # ff0000 # ff0000 0 0
Gateway Recomb4 cmastwtGTATAGAaaagywg misc_feature # ff0000 # ff0000 0 0
Gateway Recomb3 cmastwtGTATAATaaagywg misc_feature # ff0000 # ff0000 0 0
Gateway Recomb2 cmastwtGTACAAGaaagywg misc_feature # ff0000 # ff0000 0 0
Gateway recomb1 cmastwtGTACAAAaaagywg misc_feature # ff0000 # ff0000 0 0

manulera commented 4 weeks ago

Hi @BjornFJohansson thanks for sharing, where did you get those consensus sequences from? I have been talking about this with @dgruano and made some alignments from the SnapGene example plasmids, if you want to have a look it's well summarised in this README:

https://github.com/manulera/gateway_sequences

manulera commented 4 weeks ago

For implementing this the new Assembly class can be easily used, it's just a matter of defining what constitutes the consensus site of each type, which I could not find in the literature nor online documentation from suppliers.

manulera commented 3 weeks ago

@BjornFJohansson where did you get these consensual sequences? I can see that ApE ships a file with some sequences (copied below), but for instance the attP5 is not included

attB4   CMASTTTGTATAGAAAAGYWG   misc_recomb #dda0dd #dda0dd 0   0   
attB3   CMASTTTGTATAATAAAGYWG   misc_recomb #dda0dd #dda0dd 0   0   
AttB2   CMASTTTGTACAAGAAAGYWG   misc_feature    #dda0dd #dda0dd 0   0   
AttB1   CMASTTTGTACAAAAAAGYWG   misc_feature    #dda0dd #dda0dd 0   0   
attP4   AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTATAGAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATSCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA    misc_recomb #dda0dd #dda0dd 0   0   
attP3   AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTATAATAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATSCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA    misc_recomb #dda0dd #dda0dd 0   0   
AttP2   AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTACAAGAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATSCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA    misc_recomb plum    plum    0   0   
AttP1   AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTACAAAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATSCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA    misc_recomb plum    plum    0   0   
attL4   AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTATAGAAAAGYWG    misc_recomb #dda0dd #dda0dd 0   0   
attL3   AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTATAATAAAGYWG    misc_recomb #dda0dd #dda0dd 0   0   
AttL2   AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTACAAGAAAGYWG    misc_recomb plum    plum    0   0   
AttL1   AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAMATTGATRAGCAATGCTTTYTTATAATGCCMASTTTGTACAAAAAAGYWG    misc_recomb plum    plum    0   0   
attR4   CMASTTTGTATAGAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATSCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA   misc_recomb #dda0dd #dda0dd 0   0   
attR3   CMASTTTGTATAATAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATSCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA   misc_recomb #dda0dd #dda0dd 0   0   
AttR2   CMASTTTGTACAAGAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATSCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA   misc_recomb plum    plum    0   0   
AttR1   CMASTTTGTACAAAAAAGYWGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATSCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA   misc_recomb plum    plum    0   0   
ccdB    ATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCNCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATA  CDS yellow  yellow  0   0   
Gateway Recomb4 cmastttGTATAGAaaagywg   misc_feature    #ff0000 #ff0000 0   0   
Gateway Recomb3 cmastttGTATAATaaagywg   misc_feature    #ff0000 #ff0000 0   0   
Gateway Recomb2 cmastttGTACAAGaaagywg   misc_feature    #ff0000 #ff0000 0   0   
Gateway recomb1 cmastttGTACAAAaaagywg   misc_feature    #ff0000 #ff0000 0   0
BjornFJohansson commented 1 week ago

I did get those from the ApE source code.

image

The newest version (3.1.7) has even attP6