Team:Edinburgh/Sequences

From 2011.igem.org

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(Vector with PlacLacZ-INP to amplify for Gibson)
(PlacLacZ - INP - bglX (expected))
 
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==Vector with PlacLacZ-INP to amplify for Gibson==
+
== PlacLacZ - INP - bglX (expected)==
 +
 
 +
<code>
 +
<font color="blue">GAATTCGCGGCCGCTTCTAGAG</font><br><br>
 +
'''ttaatacgactcactatagggagaggaggtacgatct'''<br><br>
 +
gctggggcaaaccagcgtggacc<br>
 +
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac
 +
tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg
 +
ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc
 +
aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt
 +
ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat
 +
gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg
 +
cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga
 +
agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt
 +
tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga<br><br>
 +
'''TACTAGCtgatatttcgcgtaaggaaatccattatgGGATCT'''<br><br>
 +
atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt
 +
ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt
 +
cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt
 +
catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa
 +
gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc
 +
gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct
 +
accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct
 +
gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct
 +
gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg
 +
acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct
 +
aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca
 +
ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct
 +
aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt
 +
cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg
 +
gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa<br>
 +
gatgatgattggatcgaagtgaaa gg<br><br>
 +
'''T ACT AGC agc agc att gcg agc agc agc ccg agc agc gtg gcg GGA TCT'''<br><br>
 +
atgaaatggctatgttcagtaggaatcgcggtgagtctggccctgcaaccagcact
 +
ggcggatgatttattcggcaaccatccattaacgcccgaagcgcgggatgcgttcgtcac
 +
cgaactgcttaagaaaatgacagttgatgagaaaattggtcagctgcgcttaatcagcgt
 +
cggcccggataacccgaaagaggcgatccgcgagatgatcaaagacggtcaggttggggc
 +
gattttcaacaccgtaacccgtcaggatatccgcgccatgcaggatcaggtgatggaatt
 +
aagccgcctgaaaattcctcttttctttgcttacgacgtgctgcacggtcagcgcacggt
 +
gttcccgattagcctcggtctggcctcgtcttttaacctcgatgcagtgaaaacggtcgg
 +
acgtgtctctgcttatgaagcggcagatgatggcctgaatatgacctgggcaccgatggt
 +
cgatgtctcgcgcgatccgcgctggggacgtgcttccgaaggttttggcgaagatacgta
 +
tctcacctcaacaatgggtaaaaccatggtggaagcgatgcagggtaaaagcccggcaga
 +
tcgctactcggtgatgaccagcgtcaaacactttgccgcatacggcgcggtagaaggcgg
 +
taaagagtacaacaccgtcgatatgagtccgcagcgcctgtttaatgattatatgccgcc
 +
gtacaaagcggggctggacgcaggcagcggcgcggtgatggtggcgctgaactcgctgaa
 +
cggcacgccagccacctccgattcctggctgctgaaagatgttctgcgcgaccagtgggg
 +
ctttaaaggcatcaccgtttccgatcacggtgcaatcaaagagctgattaaacatggcac
 +
ggcggcagacccggaagatgcggtgcgcgtggcgctgaaatccggaatcaacatgagcat
 +
gagcgacgagtactactcgaagtatctgcctgggttgattaaatccggcaaagtgacgat
 +
ggcagagctggacgatgctgcccgccatgtactgaacgttaaatatgatatggggttgtt
 +
taacgacccatacagccatttggggccgaaagagtctgacccggtggataccaatgccga
 +
aagccgcctgcaccgtaaagaagcgcgtgaagtggcgcgcgaaagcttggtgttgctgaa
 +
aaaccgtctcgaaacgttaccgctgaaaaaatcggccaccattgcggtggttgggccact
 +
ggcggacagtaaacgtgacgtgatgggcagctggtccgcagccggtgttgccgatcaatc
 +
cgtgaccgtactgaccgggattaaaaatgccgtcggtgaaaacggtaaagtgctgtatgc
 +
caaaggggcgaacgttaccagtgacaaaggcattatcgatttcctgaatcagtatgaaga
 +
agcggtcaaagtcgatccgcgttcgccgcaagagatgattgatgaagcggtgcagacggc
 +
gaaacaatctgatgtggtggtggctgtagtcggtgaagcacaggggatggcgcacgaagc
 +
ctccagccggaccgatatcactattccgcaaagccaacgtgacttgattgcggcgctgaa
 +
agccaccggtaaaccgctggtgctggtgctgatgaacgggcgtccgctggcgctggtgaa
 +
agaagatcagcaggctgatgcgattctggaaacctggtttgcggggactgaaggcggtaa
 +
tgcaattgccgatgtattgtttggcgattacaacccgtccggcaagctgccaatgtcctt
 +
cccgcgttctgtcgggcagatcccggtgtactacagccatctgaataccggtcgcccgta
 +
taatgccgacaagccgaacaaatacacttcgcgttattttgatgaagctaacggggcgtt
 +
gtatccgttcggctatgggctgagctacaccactttcaccgtctctgatgtgaaactttc
 +
tgcgccgaccatgaagcgtgacggcaaagtgactgccagcgtgcaggtgacgaacaccgg
 +
taagcgcgagggtgccacggtagtgcagatgtacttgcaggatgtgactgcttccatgag
 +
tcgccctgtgaaacagctgaaaggctttgagaaaatcaccctgaaaccgggcgaaactca
 +
gactgtcagcttcccgatcgatattgaggcgctgaagttctggaatcaacagatgaaata
 +
tgacgccgagcctggcaagttcaatgtctttatcggcactgattccgcacgcgttaagaa<br>
 +
aggcgagtttgagttgctggg
 +
<br><br>
 +
'''TACTAGC aacatatcataacggagtgatcgcaatg GGATCT'''<br><br>
 +
gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt<br>
 +
tctgcgtttata
 +
</code>
 +
 
 +
== PlacLacZ - INP - cex (expected)==
 +
 
 +
<code>
 +
<font color="blue">GAATTCGCGGCCGCTTCTAGAG</font><br><br>
 +
'''ttaatacgactcactatagggagaggaggtacgatct'''<br><br>
 +
gctggggcaaaccagcgtggacc<br>
 +
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac
 +
tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg
 +
ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc
 +
aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt
 +
ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat
 +
gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg
 +
cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga
 +
agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt
 +
tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga<br><br>
 +
'''TACTAGCtgatatttcgcgtaaggaaatccattatgGGATCT'''<br><br>
 +
atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt
 +
ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt
 +
cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt
 +
catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa
 +
gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc
 +
gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct
 +
accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct
 +
gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct
 +
gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg
 +
acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct
 +
aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca
 +
ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct
 +
aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt
 +
cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg
 +
gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa<br>
 +
gatgatgattggatcgaagtgaaa gg<br><br>
 +
'''T ACT AGC agc agc att gcg agc agc agc ccg agc agc gtg gcg GGA TCT'''<br><br>
 +
atggcgaccacgctcaaggaggccgccgacggcgccggccgggacttcggcttcgcgctc
 +
gaccccaaccggctctcggaggcgcagtacaaggcgatcgccgacagcgagttcaacctc
 +
gtcgtcgccgagaacgcgatgaagtggggcgccaccgagccctcgcagaacagcttctcc
 +
ttcggcgcgggcgaccgcgtcgcgagctacgccgccgacaccggcaaggagctgtacggc
 +
cacacgctcgtctggcactcgcagctgcccgactgggcgaagaacctcaacggctccgcg
 +
ttcgagagcgcgatggtcaaccacgtgacgaaggtcgccgaccacttcgagggcaaggtc
 +
gcgtcgtgggacgtcgtcaacgaggcgttcgccgacggcggcggccgccggcaggactcg
 +
gcgttccagcagaagctcggcaacggctacatcgagaccgcgttccgggcggcacgtgcg
 +
gcggacccgaccgccaagctgtgcatcaacgactacaacgtcgagggcatcaacgcgaag
 +
agcaactcgctctacgacctcgtcaaggacttcaaggcgcgcggcgtcccgctcgactgc
 +
gtcgggttccagtcgcacctcatcgtcggccaggtgccgggcgacttccggcagaacctg
 +
caacggttcgcggacctgggcgtggacgtgcgcatcaccgagctcgacatccgcatgcgg
 +
acgccctccgacgcgaccaagctcgcgacccaggcggccgactacaagaaggtcgtgcag
 +
gcctgcatgcaggtgacccgctgccagggcgtgaccgtctggggcatcaccgacaagtac
 +
tcgtgggtgccggacgtcttcccgggcgagggggccgcgctggtgtgggacgcgagctac
 +
gccaagaagccggcctacgccgccgtgatggaggccttcggcgcgagcccgacgccgacg
 +
cccaccacgccgaccccgacgcccacgacgccgacgccgaccccgacgtccggtccggcc
 +
gggtgccaggtgctgtggggcgtcaaccagtggaacaccggcttcaccgcgaacgtcacc
 +
gtgaagaacacgtcctccgctccggtcgacggctggacgctcacgttcagcttcccgtcc
 +
ggccagcaggtcacccaggcgtggagctcgacggtcacgcagtccggctcggccgtgacg
 +
gtccgcaacgccccgtggaacggctcgatcccggcgggcggcaccgcgcagttcggcttc
 +
aacggctcgcacacgggcaccaacgccgcgccgacggcgttctcgctcaacggcacgccc<br>
 +
tgcacggtcggctc
 +
<br><br>
 +
'''TACTAGC aacatatcataacggagtgatcgcaatg GGATCT'''<br><br>
 +
gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt<br>
 +
tctgcgtttata
 +
</code>
 +
 
 +
==crtEIB (expected)==
 +
 
 +
* I have assumed spacerT7 - PlacLacZ - spacer1 - E - spacer2 - I - spacer3 - B - spacer4 - stuffing
 +
 
 +
<code>
 +
<font color="blue">GAATTCGCGGCCGCTTCTAGAG</font><br><br>
 +
'''ttaatacgactcactatagggagaggaggtacgatct'''<br><br>
 +
gctggggcaaaccagcgtggacc<br>
 +
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac
 +
tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg
 +
ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc
 +
aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt
 +
ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat
 +
gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg
 +
cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga
 +
agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt
 +
tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga<br><br>
 +
'''TACTAGCtgatatttcgcgtaaggaaatccatt'''atg'''GGATCT'''<br><br>
 +
atgacggtctgcgcaaaaaaacacgttcatctcactcgcgatgctgcggagcagttactg
 +
gctgatattgatcgacgccttgatcagttattgcccgtggagggagaacgggatgttgtg
 +
ggtgccgcgatgcgtgaaggtgcgctggcaccgggaaaacgtattcgccccatgttgctg
 +
ttgctgaccgcccgcgatctgggttgcgctgtcagccatgacggattactggatttggcc
 +
tgtgcggtggaaatggtccacgcggcttcgctgatccttgacgatatgccctgcatggac
 +
gatgcgaagctgcggcgcggacgccctaccattcattctcattacggagagcatgtggca
 +
atactggcggcggttgccttgctgagtaaagcctttggcgtaattgccgatgcagatggc
 +
ctcacgccgctggcaaaaaatcgggcggtttctgaactgtcaaacgccatcggcatgcaa
 +
ggattggttcagggtcagttcaaggatctgtctgaaggggataagccgcgcagcgctgaa
 +
gctattttgatgacgaatcactttaaaaccagcacgctgttttgtgcctccatgcagatg
 +
gcctcgattgttgcgaatgcctccagcgaagcgcgtgattgcctgcatcgtttttcactt
 +
gatcttggtcaggcatttcaactgctggacgatttgaccgatggcatgaccgacaccggt
 +
aaggatagcaatcaggacgccggtaaatcgacgctggtcaatctgttaggcccgagggcg
 +
gttgaagaacgtctgagacaacatcttcagcttgccagtgagcatctctctgcggcctgc
 +
caacacgggcacgccactcaacattttattcaggcctggtttgacaaaaaactcgctgcc<br>
 +
gtcagt gg<br><br>
 +
'''TACTAGCaacatatca'''taa'''cggagtgatcgca'''atg'''GGATCT'''<br><br>
 +
atgaaaccaactacggtaattggtgcaggcttcggtggcctggcactggcaattcgtcta
 +
caagctgcggggatccccgtcttactgcttgaacaacgtgataaacccggcggtcgggct
 +
tatgtctacgaggatcaggggtttacctttgatgcaggcccgacggttatcaccgatccc
 +
agtgccattgaagaactgtttgcactggcaggaaaacagttaaaagagtatgtcgaactg
 +
ctgccggttacgccgttttaccgcctgtgttgggagtcagggaaggtctttaattacgat
 +
aacgatcaaacccggctcgaagcgcagattcagcagtttaatccccgcgatgtcgaaggt
 +
tatcgtcagtttctggactattcacgcgcggtgtttaaagaaggctatctaaagctcggt
 +
actgtcccttttttatcgttcagagacatgcttcgcgccgcacctcaactggcgaaactg
 +
caagcatggagaagcgtttacagtaaggttgccagttacatcgaagatgaacatctgcgc
 +
caggcgttttctttccactcgctgttggtgggcggcaatcccttcgccacctcatccatt
 +
tatacgttgatacacgcgctggagcgtgagtggggcgtctggtttccgcgtggcggcacc
 +
ggcgcattagttcaggggatgataaagctgtttcaggatctgggtggcgaagtcgtgtta
 +
aacgccagagtcagccatatggaaacgacaggaaacaagattgaagccgtgcatttagag
 +
gacggtcgcaggttcctgacgcaagccgtcgcgtcaaatgcagatgtggttcatacctat
 +
cgcgacctgttaagccagcaccctgccgcggttaagcagtccaacaaactgcaaactaag
 +
cgcatgagtaactctctgtttgtgctctattttggtttgaatcaccatcatgatcagctc
 +
gcgcatcacacggtttgtttcggcccgcgttaccgcgagctgattgacgaaatttttaat
 +
catgatggcctcgcagaggacttctcactttatctgcacgcgccctgtgtcacggattcg
 +
tcactggcgcctgaaggttgcggcagttactatgtgttggcgccggtgccgcatttaggc
 +
accgcgaacctcgactggacggttgaggggccaaaactacgcgaccgtatttttgcgtac
 +
cttgagcagcattacatgcctggcttacggagtcagctggtcacgcaccggatgtttacg
 +
ccgtttgattttcgcgaccagcttaatgcctatcatggctcagccttttctgtggagccc
 +
gttcttacccagagcgcctggtttcggccgcataaccgcgataaaaccattactaatctc
 +
tacctggtcggcgcaggcacgcatcccggcgcaggcattcctggcgtcatcggctcggca<br>
 +
aaagcgacagcaggtttgatgctggaggatctgata gg<br><br>
 +
'''TACTAGC'''tga'''acgtaaatactcagtaaagaggagaaaGGATCT'''<br><br>
 +
atgaataatccgtcgttactcaatcatgcggtcgaaacgatggcagttggctcgaaaagt
 +
tttgcgacagcctcaaagttatttgatgcaaaaacccggcgcagcgtactgatgctctac
 +
gcctggtgccgccattgtgacgatgttattgacgatcagacgctgggctttcaggcccgg
 +
cagcctgccttacaaacgcccgaacaacgtctgatgcaacttgagatgaaaacgcgccag
 +
gcctatgcaggatcgcagatgcacgaaccggcgtttgcggcttttcaggaagtggctatg
 +
gctcatgatatcgccccggcttacgcgtttgatcatctggaaggcttcgccatggatgta
 +
cgcgaagcgcaatacagccaactggatgatacgctgcgctattgctatcacgttgcaggc
 +
gttgtcggcttgatgatggcgcaaatcatgggcgtgcgggataacgccacgctggaccgc
 +
gcctgtgaccttgggctggcatttcagttgaccaatattgctcgcgatattgtggacgat
 +
gcgcatgcgggccgctgttatctgccggcaagctggctggagcatgaaggtctgaacaaa
 +
gagaattatgcggcacctgaaaaccgtcaggcgctgagccgtatcgcccgtcgtttggtg
 +
caggaagcagaaccttactatttgtctgccacagccggcctggcagggttgcccctgcgt
 +
tccgcctgggcaatcgctacggcgaagcaggtttaccggaaaataggtgtcaaagttgaa
 +
caggccggtcagcaagcctgggatcagcggcagtcaacgaccacgcccgaaaaattaacg
 +
ctgctgctggccgcctctggtcaggcccttacttcccggatgcgggctcatcctccccgc<br>
 +
cctgcgcatctctggcagcgcccgctc gg<br><br>
 +
'''TACTAGC'''tag'''gtcttagtcttcgtcaaagaggggacaGGATCT'''<br><br>
 +
gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt<br>
 +
tctgcgtttata
 +
</code>
 +
 
 +
==Vector piece, with PlacLacZ-INP to amplify for Gibson==
 +
 
 +
<code>
 +
( gtcacactggctcaccttcgggtgggcctttctgcgtttata )
 +
</code>
<code>
<code>
Line 673: Line 899:
cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg
cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg
gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa<br>
gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa<br>
-
gatgatgattggatcgaagtgaaagg<br><br>
+
gatgatgattggatcgaagtgaaa<br><br>
-
'''t act agc ccg acg acc agc ccc acg ccg acc ccg'''
+
gg'''tactagcccgacgaccagc'''
</code>
</code>

Latest revision as of 23:28, 16 September 2011

Sequences Sandbox

It is often useful to have nucleotide sequences of various stuff that we use. This page serves as a sandbox for playing with sequence information.

Note that any biobricks we submit to the Registry have to be properly documented there.

We have BBa_K523000 to BBa_K523999 available this year.

Contents

Biobrick prefix, suffix, scar

  • gaattcgcggccgcttctag - prefix, short
  • gaattcgcggccgcttctagag - prefix, long
  • tactagtagcggccgctgcag - suffix
  • tactag - scar, short
  • tactagag - scar, long

Restriction sites

  • gaattc - EcoRI (prefix 1)
  • tctaga - XbaI (prefix 2)
  • actagt - SpeI (suffix 1)
  • ctgcag - PstI (suffix 2)
  • gcggccgc - NotI (in both prefix and suffix)
  • agatct - BglII (not part of RFC10, we use it internally after the prefix)
  • tacgta - SnaBI (might become relevant)

pSB1C3 vector

  • This is the official sequence from the Registry ([http://partsregistry.org/wiki/index.php?title=Part:pSB1C3 here])
  • Red is CmlR coding sequence ([http://en.wikipedia.org/wiki/Chloramphenicol_acetyltransferase chloramphenicol acetyltransferase])
  • Blue is restriction sites for the 4 main enzymes (SpeI, PstI, EcoRI, XbaI)

tactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgcccttt ttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgct gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc caggaaccgtaaaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacga gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagata ccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttac cggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctg taggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattac gcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctca gtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac ttggtctgacagctcgaggcttggattctcaccaataaaaaacgcccggcggcaaccgag cgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtcc aagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaatt cattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgcca gcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcga agaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattgg ctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgt aacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcac tccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacac tatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattca tcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacgg tctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactg actgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatc cagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaa atacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgcccga tcaactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaa aataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggat
gatttctggaattcgcggccgcttctagag

Plac-LacZα insert (clone 2)

  • Assigned code <partinfo>BBa_K523000</partinfo>
  • A modified version (seems to lack about 45 bases at the start) of [http://partsregistry.org/wiki/index.php?title=Part:BBa_J33207 BBa_J33207]
  • Sanger sequencing seemed to confirm that all was well with clone 2, though in a few regions the chromatograms were poor.
  • Sequence below includes BioBrick prefix and suffix (blue) as well as the BglII site (bold), which is partly in the prefix
  • lacZα is in red.
  • Both forward and reverse sequencing reactions failed to give good info about the suffix; so I have simply added the standard suffix. However we've not conclusively proved that the SpeI site exists; this should be verified by digest.

gaattcgcggccgcttctagagatctgctggggcaaaccagcgtggaccgcttgctgcaa ctctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaaga aaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcatta atgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaa tgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtat gttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatta cggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaac ttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgca ccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttc cggcaccagaagcggtgccggaaagctggctggagtgatactagtagcggccgctgcag

malS using primers f1/r1 (clone 1)

  • Assigned code <partinfo>BBa_K523001</partinfo>
  • Expected sequence is [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=3735520&to=3737550&report=gbwithparts derived from this].
  • Prefix and suffix in blue.
  • Pre-coding sequence in CAPITALS and contains native RBS.
  • BglII site in bold.
  • Double stop codon is present.
  • This has been sequenced and looks good.

gaattcgcggccgcttctagagatctGAAATCGCAGCAATAAGGACTCATCCGCCatgaa actcgccgcctgttttctgacactccttcctggcttcgccgttgccgccagctggacttc tccggggtttcccgcctttagcgaacaggggacaggaacatttgtcagccacgcgcagtt gcccaaaggtacgcgtccactaacgctaaattttgaccaacagtgctggcagcctgcgga tgcgataaaactcaatcagatgctttccctgcaaccttgtagcaacacgccgcctcaatg gcgattgttcagggacggcgaatatacgctgcaaatagacacccgctccggtacgccaac attgatgatttccatccagaacgccgccgaaccggtagcaagcctggtccgtgaatgccc gaaatgggatggattaccgctcacagtggatgtcagcgccactttcccggaaggagccgc cgtacgggattattacagccagcaaattgcgatagtgaagaacggtcaaataatgttaca acccgctgccaccagcaacggtttactcctgctggaacgggcagaaactgacacatccgc ccctttcgactggcataacgccacggtttactttgtgctgacagatcgtttcgaaaacgg cgatcccagtaatgaccagagttacggacgtcataaagacggtatggcggaaattggcac ttttcacggcggcgatttacgcggcctgaccaacaaactggattacctccagcagttggg cgttaatgctttatggataagcgccccatttgagcaaattcacggctgggtcggcggcgg tacaaaaggcgatttcccgcattatgcctaccacggttattacacacaggactggacgaa tcttgatgccaatatgggcaacgaagccgatctacggacgctggttgatagcgcacatca gcgcggtattcgtattctctttgatgtcgtgatgaaccacaccggctatgccacgctggc ggatatgcaggagtatcagtttggcgcgttatatctttctggtgacgaagtgaaaaaatc gctgggtgaacgctggagcgactggaaacctgccgccgggcaaacctggcatagctttaa cgattacattaatttcagcgacaaaacaggctgggataaatggtggggaaaaaactggat cagaacggatatcggcgattacgacaatcctggattcgacgatctcactatgtcgctagc ctttttgccggatatcaaaaccgaatcaactaccgcttctggtctgccggtgttctataa aaacaaaatggatacccacgccaaagccattgacggctatacgccgcgcgattacttaac ccactggttaagtcagtgggtccgcgactatgggattgatggttttcgggtcgataccgc caaacatgttgagttgcccgcctggcagcaactgaaaaccgaagccagcgccgcgcttcg cgaatggaaaaaagctaaccccgacaaagcattagatgacaaacctttctggatgaccgg tgaagcctggggccacggcgtgatgcaaagtgactactatcgccacggcttcgatgcgat gatcaatttcgattatcaggagcaggcggcgaaagcagtcgactgtctggcgcagatgga tacgacctggcagcaaatggcggagaaattgcagggtttcaacgtgttgagctacctctc gtcgcatgatacccgcctgttccgtgaagggggcgacaaagcagcagagttattactatt agcgccaggcgcggtacaaatcttttatggtgatgaatcctcgcgtccgttcggtcctac aggttctgatccgctgcaaggtacacgttcggatatgaactggcaggatgttagcggtaa atctgccgccagcgtcgcgcactggcagaaaatcagccagttccgcgcccgccatcccgc aattggcgcgggcaaacaaacgacacttttgctgaagcagggctacggctttgttcgtga gcatggcgacgataaagtgctggtcgtctgggcagggcaacagtaataatactagtagcg
gccgctgcag

malS using primers f2/r2 (clone 1)

  • Assigned code <partinfo>BBa_K523003</partinfo>
  • This is essentially as above but starting at the start codon, and with no stop codon, and adding two bases at the end of the BioBrick, which (together with the T at the start of the suffix) code for a glycine when using BioSandwich assembly.
  • Expected sequence is [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=3735520&to=3737550&report=gbwithparts derived from this].
  • This has been sequenced and looks good.

gaattcgcggccgcttctagagatctatgaaactcgccgcctgttttctgacactccttc ctggcttcgccgttgccgccagctggacttctccggggtttcccgcctttagcgaacagg ggacaggaacatttgtcagccacgcgcagttgcccaaaggtacgcgtccactaacgctaa attttgaccaacagtgctggcagcctgcggatgcgataaaactcaatcagatgctttccc tgcaaccttgtagcaacacgccgcctcaatggcgattgttcagggacggcgaatatacgc tgcaaatagacacccgctccggtacgccaacattgatgatttccatccagaacgccgccg aaccggtagcaagcctggtccgtgaatgcccgaaatgggatggattaccgctcacagtgg atgtcagcgccactttcccggaaggagccgccgtacgggattattacagccagcaaattg cgatagtgaagaacggtcaaataatgttacaacccgctgccaccagcaacggtttactcc tgctggaacgggcagaaactgacacatccgcccctttcgactggcataacgccacggttt actttgtgctgacagatcgtttcgaaaacggcgatcccagtaatgaccagagttacggac gtcataaagacggtatggcggaaattggcacttttcacggcggcgatttacgcggcctga ccaacaaactggattacctccagcagttgggcgttaatgctttatggataagcgccccat ttgagcaaattcacggctgggtcggcggcggtacaaaaggcgatttcccgcattatgcct accacggttattacacacaggactggacgaatcttgatgccaatatgggcaacgaagccg atctacggacgctggttgatagcgcacatcagcgcggtattcgtattctctttgatgtcg tgatgaaccacaccggctatgccacgctggcggatatgcaggagtatcagtttggcgcgt tatatctttctggtgacgaagtgaaaaaatcgctgggtgaacgctggagcgactggaaac ctgccgccgggcaaacctggcatagctttaacgattacattaatttcagcgacaaaacag gctgggataaatggtggggaaaaaactggatcagaacggatatcggcgattacgacaatc ctggattcgacgatctcactatgtcgctagcctttttgccggatatcaaaaccgaatcaa ctaccgcttctggtctgccggtgttctataaaaacaaaatggatacccacgccaaagcca ttgacggctatacgccgcgcgattacttaacccactggttaagtcagtgggtccgcgact atgggattgatggttttcgggtcgataccgccaaacatgttgagttgcccgcctggcagc aactgaaaaccgaagccagcgccgcgcttcgcgaatggaaaaaagctaaccccgacaaag cattagatgacaaacctttctggatgaccggtgaagcctggggccacggcgtgatgcaaa gtgactactatcgccacggcttcgatgcgatgatcaatttcgattatcaggagcaggcgg cgaaagcagtcgactgtctggcgcagatggatacgacctggcagcaaatggcggagaaat tgcagggtttcaacgtgttgagctacctctcgtcgcatgatacccgcctgttccgtgaag ggggcgacaaagcagcagagttattactattagcgccaggcgcggtacaaatcttttatg gtgatgaatcctcgcgtccgttcggtcctacaggttctgatccgctgcaaggtacacgtt cggatatgaactggcaggatgttagcggtaaatctgccgccagcgtcgcgcactggcaga aaatcagccagttccgcgcccgccatcccgcaattggcgcgggcaaacaaacgacacttt tgctgaagcagggctacggctttgttcgtgagcatggcgacgataaagtgctggtcgtct gggcagggcaacagggtactagtagcggccgctgcag

bglX using primers f1/r1 (clone 1)

  • Assigned code <partinfo>BBa_K523002</partinfo>
  • Expected sequence is [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=2217714&to=2220011&report=gbwithparts derived from this].
  • Prefix and suffix in blue.
  • Pre-coding sequence in CAPITALS and contains native RBS.
  • BglII site in bold.
  • Double stop codon is present.
  • The sequence as it stands has a PstI site (red).
  • This has been sequenced and looks good. A silent mutation from the expected sequence is indicated as a red capital T.

gaattcgcggccgcttctagagatctGCCACGTCGGGCAACAAAGGAAGAAAAATCCATa tgaaatggctatgttcagtaggaatcgcggtgagtctggccctgcagccagcactggcgg atgatttattcggcaaccatccattaacgcccgaagcgcgggatgcgttcgtcaccgaac tgcttaagaaaatgacagttgatgagaaaattggtcagctgcgcttaatcagcgtcggcc cggataacccgaaagaggcgatccgcgagatgatcaaagacggtcaggttggggcgattt tcaacaccgtaacccgtcaggatatccgcgccatgcaggatcaggtgatggaattaagcc gcctgaaaattcctcttttctttgcttacgacgtgctgcacggtcagcgcacggtgttcc cgattagcctcggtctggcctcgtcttttaacctcgatgcagtgaaaacggtcggacgtg tctctgcttatgaagcggcagatgatggcctgaatatgacctgggcaccgatggtcgatg tctcgcgcgatccgcgctggggacgtgcttccgaaggttttggcgaagatacgtatctca cctcaacaatgggtaaaaccatggtggaagcgatgcagggtaaaagcccggcagatcgct actcggtgatgaccagcgtcaaacactttgccgcatacggcgcggtagaaggcggtaaag agtacaacaccgtcgatatgagtccgcagcgcctgtttaatgattatatgccgccgtaca aagcggggctggacgcaggcagcggcgcggtgatggtggcgctgaactcgctgaacggca cgccagccacctccgattcctggctgctgaaagatgttctgcgcgaccagtggggcttta aaggcatcaccgtttccgatcacggtgcaatcaaagagctgattaaacatggcacggcgg cagacccggaagatgcggtgcgcgtggcgctgaaatccggaatcaacatgagcatgagcg acgagtactactcgaagtatctgcctgggttgattaaatccggcaaagtgacgatggcag agctggacgatgctgcccgccatgtactgaacgttaaatatgatatggggttgtttaacg acccatacagccatttggggccgaaagagtctgacccggtggataccaatgccgaaagcc gcctgcaccgtaaagaagcgcgtgaagtggcgcgcgaaagcttggtgttgctgaaaaacc gtctcgaaacgttaccgctgaaaaaatcggccaccattgcggtggttgggccactggcgg acagtaaacgtgacgtgatgggcagctggtccgcagccggtgttgccgatcaatccgtga ccgtactgaccgggattaaaaatgccgtcggtgaaaacggtaaagtgctgtatgccaaag gggcgaacgttaccagtgacaaaggcattatcgatttcctgaatcagtatgaagaagcgg tcaaagtcgatccgcgttcgccgcaagagatgattgatgaagcggtgcagacggcgaaac aatctgatgtggtggtggctgtagtcggtgaagcacaggggatggcgcacgaagcctcca gccggaccgatatcactattccgcaaagccaacgtgacttgattgcggcgctgaaagcca ccggtaaaccgctggtgctggtgctgatgaacgggcgtccgctggcgctggtgaaagaag atcagcaggctgatgcgattctggaaacctggtttgcggggactgaaggcggtaatgcaa ttgccgatgtattgtttggcgattacaacccgtccggcaagctgccaatgtccttcccgc gttctgtcgggcagatcccggtgtactacagccatctgaataccggtcgcccgtataatg ccgacaagccgaacaaatacacttcgcgttattttgatgaagctaacggggcgttgtatc cgttcggctatgggctgagctacaccactttcaccgtctctgatgtgaaactttctgcgc cgaccatgaagcgtgacggcaaagtgactgccagcgtgcaggtgacgaacaccggtaagc gcgagggtgccacggtagtgcagatgtacttgcaggatgtgacTgcttccatgagtcgcc ctgtgaaacagctgaaaggctttgagaaaatcaccctgaaaccgggcgaaactcagactg tcagcttcccgatcgatattgaggcgctgaagttctggaatcaacagatgaaatatgacg ccgagcctggcaagttcaatgtctttatcggcactgattccgcacgcgttaagaaaggcg agtttgagttgctgtaataatactagtagcggccgctgcag

bglX using primers f2/r2 (clone 1)

  • Assigned code <partinfo>BBa_K523004</partinfo>
  • This is essentially as above but starting at the start codon, and with no stop codon, and adding two bases at the end of the BioBrick, which (together with the T at the start of the suffix) code for a glycine when using BioSandwich assembly.
  • Expected sequence is [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=2217714&to=2220011&report=gbwithparts derived from this].
  • This has been sequenced and looks good. A silent mutation from the expected sequence is indicated as a red capital T.

gaattcgcggccgcttctagagatctatgaaatggctatgttcagtaggaatcgcggtga gtctggccctgcagccagcactggcggatgatttattcggcaaccatccattaacgcccg aagcgcgggatgcgttcgtcaccgaactgcttaagaaaatgacagttgatgagaaaattg gtcagctgcgcttaatcagcgtcggcccggataacccgaaagaggcgatccgcgagatga tcaaagacggtcaggttggggcgattttcaacaccgtaacccgtcaggatatccgcgcca tgcaggatcaggtgatggaattaagccgcctgaaaattcctcttttctttgcttacgacg tgctgcacggtcagcgcacggtgttcccgattagcctcggtctggcctcgtcttttaacc tcgatgcagtgaaaacggtcggacgtgtctctgcttatgaagcggcagatgatggcctga atatgacctgggcaccgatggtcgatgtctcgcgcgatccgcgctggggacgtgcttccg aaggttttggcgaagatacgtatctcacctcaacaatgggtaaaaccatggtggaagcga tgcagggtaaaagcccggcagatcgctactcggtgatgaccagcgtcaaacactttgccg catacggcgcggtagaaggcggtaaagagtacaacaccgtcgatatgagtccgcagcgcc tgtttaatgattatatgccgccgtacaaagcggggctggacgcaggcagcggcgcggtga tggtggcgctgaactcgctgaacggcacgccagccacctccgattcctggctgctgaaag atgttctgcgcgaccagtggggctttaaaggcatcaccgtttccgatcacggtgcaatca aagagctgattaaacatggcacggcggcagacccggaagatgcggtgcgcgtggcgctga aatccggaatcaacatgagcatgagcgacgagtactactcgaagtatctgcctgggttga ttaaatccggcaaagtgacgatggcagagctggacgatgctgcccgccatgtactgaacg ttaaatatgatatggggttgtttaacgacccatacagccatttggggccgaaagagtctg acccggtggataccaatgccgaaagccgcctgcaccgtaaagaagcgcgtgaagtggcgc gcgaaagcttggtgttgctgaaaaaccgtctcgaaacgttaccgctgaaaaaatcggcca ccattgcggtggttgggccactggcggacagtaaacgtgacgtgatgggcagctggtccg cagccggtgttgccgatcaatccgtgaccgtactgaccgggattaaaaatgccgtcggtg aaaacggtaaagtgctgtatgccaaaggggcgaacgttaccagtgacaaaggcattatcg atttcctgaatcagtatgaagaagcggtcaaagtcgatccgcgttcgccgcaagagatga ttgatgaagcggtgcagacggcgaaacaatctgatgtggtggtggctgtagtcggtgaag cacaggggatggcgcacgaagcctccagccggaccgatatcactattccgcaaagccaac gtgacttgattgcggcgctgaaagccaccggtaaaccgctggtgctggtgctgatgaacg ggcgtccgctggcgctggtgaaagaagatcagcaggctgatgcgattctggaaacctggt ttgcggggactgaaggcggtaatgcaattgccgatgtattgtttggcgattacaacccgt ccggcaagctgccaatgtccttcccgcgttctgtcgggcagatcccggtgtactacagcc atctgaataccggtcgcccgtataatgccgacaagccgaacaaatacacttcgcgttatt ttgatgaagctaacggggcgttgtatccgttcggctatgggctgagctacaccactttca ccgtctctgatgtgaaactttctgcgccgaccatgaagcgtgacggcaaagtgactgcca gcgtgcaggtgacgaacaccggtaagcgcgagggtgccacggtagtgcagatgtacttgc aggatgtgacTgcttccatgagtcgccctgtgaaacagctgaaaggctttgagaaaatca ccctgaaaccgggcgaaactcagactgtcagcttcccgatcgatattgaggcgctgaagt tctggaatcaacagatgaaatatgacgccgagcctggcaagttcaatgtctttatcggca ctgattccgcacgcgttaagaaaggcgagtttgagttgctgggtactagtagcggccgct
gcag

INP in RFC12 format (clone 2)

  • This is <partinfo>BBa_K265008</partinfo>.
  • This came on pSB1AK3 and with an [http://dspace.mit.edu/bitstream/handle/1721.1/45139/BBFRFC12.txt?sequence=1 RFC12] prefix and suffix (capitals).
  • This has been sequenced and the thing we have is perfect except it has an extra C base in the suffix (red).
  • (Blue) There is a SpeI site in the prefix (not suffix) and a NheI site in the suffix.

GAATTCGCGGCCGCACTAGTatgaccctggataaagcgctggtgctgcgcacttgtgcca ataatatggccgaccattgtggcctgatctggcctgcttctggtacagttgaaagccgct attggcagtctacacgtcgtcacgaaaacgggctggttgggctgctgtggggtgccggaa catccgcctttctgagcgttcatgccgatgctcgttggattgtttgtgaggttgctgttg ccgacatcattagcctggaagaaccgggtatggtgaaatttcctcgtgctgaagtggtcc atgttggtgatcgtattagcgcctcccatttcatttcagctcgtcaggctgatccagcaa gcacatcaacaagcacctctaccagtacactgacaccaatgccaaccgctatccctactc ctatgccggctgtagcctctgttactctgcctgtggccgaacaagctcgccacgaagtct ttgacgttgcctctgtgtctgctgctgctgctcctgttaacactctgcctgttaccacgc ctcaaaatctgcaaacggcgacctatggctcaacactgagtggtgataaccattctcgtc tgattgccgggtatggctctaacgaaaccgccggaaaccattctgatctgatcggtggtc acgattgtacgctgatggcaggtgatcaatctcgtctgaccgctggcaaaaattccgttc tgaccgctggggcacgttctaaactgatcgggagcgaaggctctactctgtccgccggag aagattctaccctgatctttcgtctgtgggatggtaaacgttatcgtcagctggtcgctc gtacaggtgaaaatggtgtggaagccgacattccgtattatgtgaatgaggacgatgata ttgtggacaaaccggacgaagatgatgattggatcgaagtgaaaGCTAGCGCGGCCGCcT
GCAG

pVIII

  • Blue is the leader sequence... MKKSLVLKASVAVATLVPMLSFA
  • Expected sequence is [http://www.ncbi.nlm.nih.gov/nuccore/56713234?report=genbank derived from this].

atgaaaaagtctttagtcctcaaagcctctgtagccgttgctaccctcgttccgatgctg tctttcgctgctgagggtgacgatcccgcaaaagcggcctttaactccctgcaagcctca gcgaccgaatatatcggttatgcgtgggcgatggttgttgtcattgtcggcgcaactatc ggtatcaagctgtttaagaaattcacctcgaaagcaagctga

C. fimi exoglucanase (clone 2)

  • This has been sequenced.
  • Two bases are not what they should be...

atctatggcgaccacgctcaaggaggccgccgacggcgccggccgggacttcggcttcgc gctcgaccccaaccggctctcggaggcgcagtacaaggcgatcgccgacagcgagttcaa cctcgtcgtcgccgagaacgcgatgaagtgggGcgccaccgagccctcgcagaacagctt ctccttcggcgcgggcgaccgcgtcgcgagctacgccgccgacaccggcaaggagctgta cggccacacgctcgtCtggcactcgcagctgcccgactgggcgaagaacctcaacggctc cgcgttcgagagcgcgatggtcaaccacgtgacgaaggtcgccgaccacttcgagggcaa ggtcgcgtcgtgggacgtcgtcaacgaggcgttcgccgacggcggcggccgccggcagga ctcggcgttccagcagaagctcggcaacggctacatcgagaccgcgttccgggcggcacg tgcggcggacccgaccgccaagctgtgcatcaacgactacaacgtcgagggcatcaacgc gaagagcaactcgctctacgacctcgtcaaggacttcaaggcgcgcggcgtcccgctcga ctgcgtcgggttccagtcgcacctcatcgtcggccaggtgccgggcgacttccggcagaa cctgcaacggttcgcggacctgggcgtggacgtgcgcatcaccgagctcgacatccgcat gcggacgccctccgacgcgaccaagctcgcgacccaggcggccgactacaagaaggtcgt gcaggcctgcatgcaggtgacccgctgccagggcgtgaccgtctggggcatcaccgacaa gtactcgtgggtgccggacgtcttcccgggcgagggggccgcgctggtgtgggacgcgag ctacgccaagaagccggcctacgccgccgtgatggaggccttcggcgcgagcccgacgcc gacgcccaccacgccgaccccgacgcccacgacgccgacgccgaccccgacgtccggtcc ggccgggtgccaggtgctgtggggcgtcaaccagtggaacaccggcttcaccgcgaacgt caccgtgaagaacacgtcctccgctccggtcgacggctggacgctcacgttcagcttccc gtccggccagcaggtcacccaggcgtggagctcgacggtcacgcagtccggctcggccgt gacggtccgcaacgccccgtggaacggctcgatcccggcgggcggcaccgcgcagttcgg cttcaacggctcgcacacgggcaccaacgccgcgccgacggcgttctcgctcaacggcac
gccctgcacggtcggctc

pG8SAET

  • The sequence is found [http://www.ncbi.nlm.nih.gov/nuccore/AF130864.1 here].
  • The plasmid encodes a beta-lactamase (bold) for ampicillin resistance.
  • Restriction sites that exist are underlined:
    • NotI (c|catgg)
    • SnaBI (tac|gta)
  • The leader sequence (green) is derived from [http://www.ncbi.nlm.nih.gov/nuccore/EF455822.1 this].
  • The E-tag (GAPVPYPDPLEPR, blue) and pVIII sequence (red) are out of frame with the leader sequence.
  • Correct this by adding one base at the start of our inserted gene.
  • WAIT! There seems to be a stop codon between the E-tag and the pVIII sequence, how can that be?

agcggaagagccggttttaagccttttacttcctgaataaatctttcagcaaaatattta ttttataagttgtaaaacttacctttaaatttaattataaatatagattttagtattgca atacataattcgttatattatgatgactttacaaatacatacagggggtattaatttgaa aaggaaaaacatttattcaattcgtaaactaggtgtaggtattgcatctgtaactttagg tacattacttatatctggtggcgtaacacctgctgcaaatgctgcgcaacacgatgacca tggcagtacgtacccggtgcgccggtgccgtatccggacccactggaaccgcgtgcctag ggatccgagggtgacgatcccgcaaaagcggcctttgactccctgcaagcctcagcgacc gaatatatcggttatgcgtgggcgatggttgttgtcattgtcggcgcaactatcggtatc aagctgtttaagaaattcacctcgaaagcaagctgataaaccgatgaattcactggccgt cgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagc acatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttccca acagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatct gtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgca ttaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcccta gcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgt caagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtt tttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactgga acaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcg gcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaata ttaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagtta agccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccg gcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttca ccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggtt aatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgc ggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttc cgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaa acgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaa ctggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaa gagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtc acagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataacc atgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagcta accgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaata gactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggc tggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagca ctggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgg taactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaa tttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgt gagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagat cctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtg gtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga gcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaac tctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagt ggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcag cggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacacc gaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag gcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcca gggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgt cgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcc tttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcc cctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagc
cgaacgaccgagcgcagcgagtcagtgagcgagga

pSB1C3 for BioSandwich

  • Chris's primers used on pSB1C3—<partinfo>BBa_J04450</partinfo> produced this linear product.
    • Forward primer: atcagatctgttgtttgtcggtgaacg
    • Reverse primer: gagaggcggtttgcgtattg
  • Standard suffix and prefix in bold.
  • BglII and XbaI sites in red.

atcagatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgg gtgggcctttctgcgtttatatactagtagcggccgctgcagtccggcaaaaaagggcaa ggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggc ttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctca ctcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtg agcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcca caggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaa cccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcc tgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggc gctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagct gggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcg tcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacag gattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta cggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcgg aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttt tgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctt ttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgag attatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaat ctaaagtatatatgagtaaacttggtctgacagctcgaggcttggattctcaccaataaa aaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattac tggatctatcaacaggagtccaagcgagctcgatatcaaattacgccccgccctgccact catcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacaaacgg catgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgc ccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactgg tgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttaggga aataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgcc ggaaatcgtcgtggtattcactccagagcgatgaaaacgtttcagtttgctcatggaaaa cggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatac gaaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaact tgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggt tataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattggg atatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctg aaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagt tggaacctcttacgtgcccgatcaactcgagtgccacctgacgtctaagaaaccattatt atcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaa tccttagctttcgctaaggatgatttctggaattcgcggccgcttctagagcaatacgca
aaccgcctctc

BioSandwich test with PlacLacZ and EYFP

  • Expected sequence...
  • Bold is the chosen parts of the spacers...

ttaatacgactcactatagggagaggaggtacgatctgctggggcaaaccagcgtggacc gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgatactagctgat atttcgcgtaaggaaatccattatgggatctatggtgagcaagggcgaggagctgttcac cggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgt gtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcac caccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgca atgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcc cgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccg cgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcga cttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaa cgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgcca caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcgg cgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaa agaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggat cactctcggcatggacgagctgtacaagtaataatactagcaacatatcataacggagtg atcgcaatgggatctgttgtttgtcggtgaacgctctctactagagtcacactggctcac
cttcgggtgggcctttctgcgtttata

Alternate BioSandwich test with PlacLacZ and EYFP

  • Chris did a different version of OEPCR with different primers and a blunt end ligation.
  • This gave a different sequence, lacking spacer2 and having an RFC54 suffix before the end of the part.

ttaatacgactcactatagggagaggaggtacgatctgctggggcaaaccagcgtggacc gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgatactagctgat atttcgcgtaaggaaatccattatgggatctatggtgagcaagggcgaggagctgttcac cggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgt gtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcac caccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgca atgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcc cgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccg cgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcga cttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaa cgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgcca caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcgg cgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaa agaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggat cactctcggcatggacgagctgtacaagtaataatactagtagctgcagacaatcagatc tgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcct
ttctgcgtttata


Serendipitous PlacLacZ-INP construct

  • While attempting to make PlacLacZ-INP-malS, we got the following...
  • malS is not present
  • But it will be useful as a template for true Gibson Assembly.


GAATTCGCGGCCGCTTCTAGAG

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

tactagctgatatttcgcgtaaggaaatccattatgGGATCT

atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa
gatgatgattggatcgaagtgaaagg

t act agc ccg acg acc agc ccc acg ccg acc ccg

Sequence is not as expected after this. Note also repetitive sequences that might interfere with primer design.

PlacLacZ - INP - YFP (expected)

GAATTCGCGGCCGCTTCTAGAG

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

TACTAGCtgatatttcgcgtaaggaaatccattatgGGATCT

atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa
gatgatgattggatcgaagtgaaa gg

T ACT AGC ccg acg acc agc ccc acg ccg acc ccg acg GGA TCT

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggac ggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctac ggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccacc ctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaag ctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttc ttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctg gtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcac aagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaac ggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgcc gaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccac tacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtc ctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa
taa

TACTAGC aacatatcataacggagtgatcgcaatg GGATCT

gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt
tctgcgtttata


PlacLacZ - INP - bglX (expected)

GAATTCGCGGCCGCTTCTAGAG

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

TACTAGCtgatatttcgcgtaaggaaatccattatgGGATCT

atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa
gatgatgattggatcgaagtgaaa gg

T ACT AGC agc agc att gcg agc agc agc ccg agc agc gtg gcg GGA TCT

atgaaatggctatgttcagtaggaatcgcggtgagtctggccctgcaaccagcact ggcggatgatttattcggcaaccatccattaacgcccgaagcgcgggatgcgttcgtcac cgaactgcttaagaaaatgacagttgatgagaaaattggtcagctgcgcttaatcagcgt cggcccggataacccgaaagaggcgatccgcgagatgatcaaagacggtcaggttggggc gattttcaacaccgtaacccgtcaggatatccgcgccatgcaggatcaggtgatggaatt aagccgcctgaaaattcctcttttctttgcttacgacgtgctgcacggtcagcgcacggt gttcccgattagcctcggtctggcctcgtcttttaacctcgatgcagtgaaaacggtcgg acgtgtctctgcttatgaagcggcagatgatggcctgaatatgacctgggcaccgatggt cgatgtctcgcgcgatccgcgctggggacgtgcttccgaaggttttggcgaagatacgta tctcacctcaacaatgggtaaaaccatggtggaagcgatgcagggtaaaagcccggcaga tcgctactcggtgatgaccagcgtcaaacactttgccgcatacggcgcggtagaaggcgg taaagagtacaacaccgtcgatatgagtccgcagcgcctgtttaatgattatatgccgcc gtacaaagcggggctggacgcaggcagcggcgcggtgatggtggcgctgaactcgctgaa cggcacgccagccacctccgattcctggctgctgaaagatgttctgcgcgaccagtgggg ctttaaaggcatcaccgtttccgatcacggtgcaatcaaagagctgattaaacatggcac ggcggcagacccggaagatgcggtgcgcgtggcgctgaaatccggaatcaacatgagcat gagcgacgagtactactcgaagtatctgcctgggttgattaaatccggcaaagtgacgat ggcagagctggacgatgctgcccgccatgtactgaacgttaaatatgatatggggttgtt taacgacccatacagccatttggggccgaaagagtctgacccggtggataccaatgccga aagccgcctgcaccgtaaagaagcgcgtgaagtggcgcgcgaaagcttggtgttgctgaa aaaccgtctcgaaacgttaccgctgaaaaaatcggccaccattgcggtggttgggccact ggcggacagtaaacgtgacgtgatgggcagctggtccgcagccggtgttgccgatcaatc cgtgaccgtactgaccgggattaaaaatgccgtcggtgaaaacggtaaagtgctgtatgc caaaggggcgaacgttaccagtgacaaaggcattatcgatttcctgaatcagtatgaaga agcggtcaaagtcgatccgcgttcgccgcaagagatgattgatgaagcggtgcagacggc gaaacaatctgatgtggtggtggctgtagtcggtgaagcacaggggatggcgcacgaagc ctccagccggaccgatatcactattccgcaaagccaacgtgacttgattgcggcgctgaa agccaccggtaaaccgctggtgctggtgctgatgaacgggcgtccgctggcgctggtgaa agaagatcagcaggctgatgcgattctggaaacctggtttgcggggactgaaggcggtaa tgcaattgccgatgtattgtttggcgattacaacccgtccggcaagctgccaatgtcctt cccgcgttctgtcgggcagatcccggtgtactacagccatctgaataccggtcgcccgta taatgccgacaagccgaacaaatacacttcgcgttattttgatgaagctaacggggcgtt gtatccgttcggctatgggctgagctacaccactttcaccgtctctgatgtgaaactttc tgcgccgaccatgaagcgtgacggcaaagtgactgccagcgtgcaggtgacgaacaccgg taagcgcgagggtgccacggtagtgcagatgtacttgcaggatgtgactgcttccatgag tcgccctgtgaaacagctgaaaggctttgagaaaatcaccctgaaaccgggcgaaactca gactgtcagcttcccgatcgatattgaggcgctgaagttctggaatcaacagatgaaata tgacgccgagcctggcaagttcaatgtctttatcggcactgattccgcacgcgttaagaa
aggcgagtttgagttgctggg

TACTAGC aacatatcataacggagtgatcgcaatg GGATCT

gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt
tctgcgtttata

PlacLacZ - INP - cex (expected)

GAATTCGCGGCCGCTTCTAGAG

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

TACTAGCtgatatttcgcgtaaggaaatccattatgGGATCT

atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa
gatgatgattggatcgaagtgaaa gg

T ACT AGC agc agc att gcg agc agc agc ccg agc agc gtg gcg GGA TCT

atggcgaccacgctcaaggaggccgccgacggcgccggccgggacttcggcttcgcgctc gaccccaaccggctctcggaggcgcagtacaaggcgatcgccgacagcgagttcaacctc gtcgtcgccgagaacgcgatgaagtggggcgccaccgagccctcgcagaacagcttctcc ttcggcgcgggcgaccgcgtcgcgagctacgccgccgacaccggcaaggagctgtacggc cacacgctcgtctggcactcgcagctgcccgactgggcgaagaacctcaacggctccgcg ttcgagagcgcgatggtcaaccacgtgacgaaggtcgccgaccacttcgagggcaaggtc gcgtcgtgggacgtcgtcaacgaggcgttcgccgacggcggcggccgccggcaggactcg gcgttccagcagaagctcggcaacggctacatcgagaccgcgttccgggcggcacgtgcg gcggacccgaccgccaagctgtgcatcaacgactacaacgtcgagggcatcaacgcgaag agcaactcgctctacgacctcgtcaaggacttcaaggcgcgcggcgtcccgctcgactgc gtcgggttccagtcgcacctcatcgtcggccaggtgccgggcgacttccggcagaacctg caacggttcgcggacctgggcgtggacgtgcgcatcaccgagctcgacatccgcatgcgg acgccctccgacgcgaccaagctcgcgacccaggcggccgactacaagaaggtcgtgcag gcctgcatgcaggtgacccgctgccagggcgtgaccgtctggggcatcaccgacaagtac tcgtgggtgccggacgtcttcccgggcgagggggccgcgctggtgtgggacgcgagctac gccaagaagccggcctacgccgccgtgatggaggccttcggcgcgagcccgacgccgacg cccaccacgccgaccccgacgcccacgacgccgacgccgaccccgacgtccggtccggcc gggtgccaggtgctgtggggcgtcaaccagtggaacaccggcttcaccgcgaacgtcacc gtgaagaacacgtcctccgctccggtcgacggctggacgctcacgttcagcttcccgtcc ggccagcaggtcacccaggcgtggagctcgacggtcacgcagtccggctcggccgtgacg gtccgcaacgccccgtggaacggctcgatcccggcgggcggcaccgcgcagttcggcttc aacggctcgcacacgggcaccaacgccgcgccgacggcgttctcgctcaacggcacgccc
tgcacggtcggctc

TACTAGC aacatatcataacggagtgatcgcaatg GGATCT

gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt
tctgcgtttata

crtEIB (expected)

  • I have assumed spacerT7 - PlacLacZ - spacer1 - E - spacer2 - I - spacer3 - B - spacer4 - stuffing

GAATTCGCGGCCGCTTCTAGAG

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

TACTAGCtgatatttcgcgtaaggaaatccattatgGGATCT

atgacggtctgcgcaaaaaaacacgttcatctcactcgcgatgctgcggagcagttactg gctgatattgatcgacgccttgatcagttattgcccgtggagggagaacgggatgttgtg ggtgccgcgatgcgtgaaggtgcgctggcaccgggaaaacgtattcgccccatgttgctg ttgctgaccgcccgcgatctgggttgcgctgtcagccatgacggattactggatttggcc tgtgcggtggaaatggtccacgcggcttcgctgatccttgacgatatgccctgcatggac gatgcgaagctgcggcgcggacgccctaccattcattctcattacggagagcatgtggca atactggcggcggttgccttgctgagtaaagcctttggcgtaattgccgatgcagatggc ctcacgccgctggcaaaaaatcgggcggtttctgaactgtcaaacgccatcggcatgcaa ggattggttcagggtcagttcaaggatctgtctgaaggggataagccgcgcagcgctgaa gctattttgatgacgaatcactttaaaaccagcacgctgttttgtgcctccatgcagatg gcctcgattgttgcgaatgcctccagcgaagcgcgtgattgcctgcatcgtttttcactt gatcttggtcaggcatttcaactgctggacgatttgaccgatggcatgaccgacaccggt aaggatagcaatcaggacgccggtaaatcgacgctggtcaatctgttaggcccgagggcg gttgaagaacgtctgagacaacatcttcagcttgccagtgagcatctctctgcggcctgc caacacgggcacgccactcaacattttattcaggcctggtttgacaaaaaactcgctgcc
gtcagt gg

TACTAGCaacatatcataacggagtgatcgcaatgGGATCT

atgaaaccaactacggtaattggtgcaggcttcggtggcctggcactggcaattcgtcta caagctgcggggatccccgtcttactgcttgaacaacgtgataaacccggcggtcgggct tatgtctacgaggatcaggggtttacctttgatgcaggcccgacggttatcaccgatccc agtgccattgaagaactgtttgcactggcaggaaaacagttaaaagagtatgtcgaactg ctgccggttacgccgttttaccgcctgtgttgggagtcagggaaggtctttaattacgat aacgatcaaacccggctcgaagcgcagattcagcagtttaatccccgcgatgtcgaaggt tatcgtcagtttctggactattcacgcgcggtgtttaaagaaggctatctaaagctcggt actgtcccttttttatcgttcagagacatgcttcgcgccgcacctcaactggcgaaactg caagcatggagaagcgtttacagtaaggttgccagttacatcgaagatgaacatctgcgc caggcgttttctttccactcgctgttggtgggcggcaatcccttcgccacctcatccatt tatacgttgatacacgcgctggagcgtgagtggggcgtctggtttccgcgtggcggcacc ggcgcattagttcaggggatgataaagctgtttcaggatctgggtggcgaagtcgtgtta aacgccagagtcagccatatggaaacgacaggaaacaagattgaagccgtgcatttagag gacggtcgcaggttcctgacgcaagccgtcgcgtcaaatgcagatgtggttcatacctat cgcgacctgttaagccagcaccctgccgcggttaagcagtccaacaaactgcaaactaag cgcatgagtaactctctgtttgtgctctattttggtttgaatcaccatcatgatcagctc gcgcatcacacggtttgtttcggcccgcgttaccgcgagctgattgacgaaatttttaat catgatggcctcgcagaggacttctcactttatctgcacgcgccctgtgtcacggattcg tcactggcgcctgaaggttgcggcagttactatgtgttggcgccggtgccgcatttaggc accgcgaacctcgactggacggttgaggggccaaaactacgcgaccgtatttttgcgtac cttgagcagcattacatgcctggcttacggagtcagctggtcacgcaccggatgtttacg ccgtttgattttcgcgaccagcttaatgcctatcatggctcagccttttctgtggagccc gttcttacccagagcgcctggtttcggccgcataaccgcgataaaaccattactaatctc tacctggtcggcgcaggcacgcatcccggcgcaggcattcctggcgtcatcggctcggca
aaagcgacagcaggtttgatgctggaggatctgata gg

TACTAGCtgaacgtaaatactcagtaaagaggagaaaGGATCT

atgaataatccgtcgttactcaatcatgcggtcgaaacgatggcagttggctcgaaaagt tttgcgacagcctcaaagttatttgatgcaaaaacccggcgcagcgtactgatgctctac gcctggtgccgccattgtgacgatgttattgacgatcagacgctgggctttcaggcccgg cagcctgccttacaaacgcccgaacaacgtctgatgcaacttgagatgaaaacgcgccag gcctatgcaggatcgcagatgcacgaaccggcgtttgcggcttttcaggaagtggctatg gctcatgatatcgccccggcttacgcgtttgatcatctggaaggcttcgccatggatgta cgcgaagcgcaatacagccaactggatgatacgctgcgctattgctatcacgttgcaggc gttgtcggcttgatgatggcgcaaatcatgggcgtgcgggataacgccacgctggaccgc gcctgtgaccttgggctggcatttcagttgaccaatattgctcgcgatattgtggacgat gcgcatgcgggccgctgttatctgccggcaagctggctggagcatgaaggtctgaacaaa gagaattatgcggcacctgaaaaccgtcaggcgctgagccgtatcgcccgtcgtttggtg caggaagcagaaccttactatttgtctgccacagccggcctggcagggttgcccctgcgt tccgcctgggcaatcgctacggcgaagcaggtttaccggaaaataggtgtcaaagttgaa caggccggtcagcaagcctgggatcagcggcagtcaacgaccacgcccgaaaaattaacg ctgctgctggccgcctctggtcaggcccttacttcccggatgcgggctcatcctccccgc
cctgcgcatctctggcagcgcccgctc gg

TACTAGCtaggtcttagtcttcgtcaaagaggggacaGGATCT

gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt
tctgcgtttata

Vector piece, with PlacLacZ-INP to amplify for Gibson

( gtcacactggctcaccttcgggtgggcctttctgcgtttata )

tactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgcccttt ttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgct gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc caggaaccgtaaaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacga gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagata ccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttac cggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctg taggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattac gcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctca gtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac ttggtctgacagctcgaggcttggattctcaccaataaaaaacgcccggcggcaaccgag cgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtcc aagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaatt cattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgcca gcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcga agaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattgg ctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgt aacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcac tccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacac tatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattca tcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacgg tctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactg actgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatc cagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaa atacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgcccga tcaactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaa aataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggat
gatttctggaattcgcggccgcttctagag

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

tactagctgatatttcgcgtaaggaaatccattatgGGATCT

atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa
gatgatgattggatcgaagtgaaa

ggtactagcccgacgaccagc