BBa_K143065
1
Aad9-T
Spectinomycin Resistance Protein (Aad9) - Terminator
2008-10-08T11:00:00Z
2015-05-08T01:10:24Z
Aad9 was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick iwth the double terminator was taken from the registry
Aad9 spectinomycin resistance protein(<bbpart>BBa_K143031</bbpart>) coupled to the double terminator (<bbpart>BBa_B0015</bbpart>).
Aad9 confers resistance to spectinomycin.
The double terminator is the most commonly used terminator and is a combination of parts <bbpart>BBa_B0010</bbpart> and <bbpart>BBa_B0012</bbpart>.
The double terminator allows the Spectinomycin resistance gene to be incorporated into a closed transcriptional unit.
false
false
_199_
0
3475
9
It's complicated
false
Aad9 was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase. The double terminator is the most commonly used registry termiantor.
false
Chris Hirst and Imperial iGEM 08
component1985266
1
BBa_B0010
component1985265
1
BBa_K143031
component1985268
1
BBa_B0012
annotation1985268
1
BBa_B0012
range1985268
1
868
908
annotation1985266
1
BBa_B0010
range1985266
1
780
859
annotation1985265
1
BBa_K143031
range1985265
1
1
771
BBa_B0010
1
BBa_B0010
T1 from E. coli rrnB
2003-11-19T12:00:00Z
2015-08-31T04:07:20Z
Transcriptional terminator consisting of a 64 bp stem-loop.
false
false
_1_
0
24
7
In stock
false
true
Randy Rettberg
annotation4184
1
stem_loop
range4184
1
12
55
annotation7018
1
BBa_B0010
range7018
1
1
80
BBa_K143012
1
Pveg
Promoter veg a constitutive promoter for B. subtilis
2008-09-10T11:00:00Z
2015-05-08T01:10:23Z
The Pveg promoter was suggested to us by Dr. Jan-Willem Veening of Newcastle University. This sequence supplied was compared to that of the DBTBS database<cite>#3</cite> then a section containing the binding site synthesised by Geneart.
Released HQ 2013
Pveg is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. Pveg contains binding sites for the ''B.sutbilis'' major sigma factor<cite>#1</cite>. Pveg in ''B.subtilis'' utilises two binding sites to cause high expression of genes<cite>#2</cite>, however our Pveg is lacking the upstream site to give a medium level of gene expression. It has been noted that the sporulation master regulatoion factor spoOA interacts with Pveg though it is not known how<cite>#3</cite>. The context with which we used the promoter Pveg is as a '''Polymerase Per Second''' (PoPS) generator.
false
true
_199_
0
2090
9
In stock
false
The biobrick part was designed to include a single binding site for the ''B.subtilis major sigma factor. In addition the biobrick standard was applied to the promoter Pveg sequence.
false
James Chappell
annotation1975704
1
Sigma A-35
range1975704
1
63
68
annotation1975705
1
Sigma A -10
range1975705
1
86
91
BBa_K143009
1
pyrD 3 IS
3??? Integration Sequence for the pyrD locus of B. subtilis
2008-09-16T11:00:00Z
2015-05-08T01:10:23Z
The 3??? integration sequence was taken from the B.subtilis chromosome and is homologous to the back section of the PyrD gene. It was synthesised by Geneart.
Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 3' integration sequence can be added to the back of a Biobrick construct and the 5' integration sequence specific for this locus (BBa_K143008) to the front of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis.
The PyrD gene has been a target for numerous integration vectors, including the shuttle vectors pPyr-Cm (GenBank Accession number AY464558) and pPyr-Kan (GenBank Accession number AY464559) [1].
Integration into the PyrD locus makes the B.subtilis auxotrophs for uracil and transformants require about 40ug/ml to allow for growth. This allows us to assay for integration by growing a replica plate with no supplemented uracil to negatively select for transformants.
false
false
_199_
0
2090
9
It's complicated
false
The PyrD integration sequences was designed from the PyrD gene's Genbank entry[1] and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and EpsE gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined.
false
James Chappell
annotation1977517
1
PyrD 3' Integration Sequence
range1977517
1
1
467
BBa_K143008
1
pyrD 5 IS
5??? Integration Sequence for the pyrD locus of B. subtilis
2008-09-15T11:00:00Z
2015-05-08T01:10:23Z
The 5??? integration sequence was taken from the B.subtilis chromosome and is homologous to the chromosome from a few hundred bp upstream of the gene's start codon until 300 bp into the gene. The part was synthesised by Geneart.
Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (<bbpart> BBa_K143004</bbpart>) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis.
The PyrD gene has been a target for numerous integration vectors, including the shuttle vectors pPyr-Cm (GenBank Accession number AY464558) and pPyr-Kan (GenBank Accession number AY464559) <cite>1</cite>.
Integration into the PyrD locus makes the ''B.subtilis'' auxotrophs for uracil and transformants require about 40ug/ml to allow for growth. This allows us to assay for integration by growing a replica plate with no supplemented uracil to negatively select for transformants.
false
false
_199_
0
2090
9
It's complicated
false
The PyrD integration sequences were designed from the PyrD gene's Genbank entry<cite>#1</cite> and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and PyrD gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined.
false
James Chappell
annotation1977518
1
PyrD 5' Integration Sequence
range1977518
1
1
468
BBa_K316020
1
BBa_K316020
B. subtilis genome integration vector, targets pyrD locus
2010-10-22T11:00:00Z
2015-05-08T01:11:56Z
false
false
_440_
0
7480
9
It's complicated
false
false
IC 2010 Team
component2098337
1
BBa_K143009
component2098321
1
BBa_K143053
component2098314
1
BBa_K143008
component2098315
1
BBa_K316002
component2098330
1
BBa_K143065
component2098335
1
BBa_K316014
annotation2098315
1
BBa_K316002
range2098315
1
477
504
annotation2098330
1
BBa_K143065
range2098330
1
636
1543
annotation2098314
1
BBa_K143008
range2098314
1
1
468
annotation2098321
1
BBa_K143053
range2098321
1
513
629
annotation2098337
1
BBa_K143009
range2098337
1
1604
2070
annotation2098335
1
BBa_K316014
range2098335
1
1552
1595
BBa_B0012
1
BBa_B0012
TE from coliphageT7
2003-01-31T12:00:00Z
2015-08-31T04:07:20Z
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Released HQ 2013
Transcription terminator for the <i>E.coli</i> RNA polymerase.
false
false
_1_
0
24
7
In stock
false
<P> <P>Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG).<P> Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
false
Reshma Shetty
annotation1690
1
polya
range1690
1
28
41
annotation1686
1
T7 TE
range1686
1
8
27
annotation1687
1
stop
range1687
1
34
34
annotation7020
1
BBa_B0012
range7020
1
1
41
BBa_K316014
1
BBa_K316014
Dif sequence followed by PmeI recognition site
2010-10-22T11:00:00Z
2015-05-08T01:11:56Z
Oligonucleotide synthesis of single stranded primers.
This composite part of <bbpart>BBa_K143000</bbpart> and <bbpart>BBa_K143013</bbpart>. The dif site can be used in conjunction with another dif site in another part of the vector to remove a sequence between the two dif sites. PmeI site can be used for blunt end cloning of a DNA sequence behind the dif site.
Please see ???Part Design??? section for design considerations and parts used.
false
false
_440_
0
7480
9
It's complicated
false
This part was designed to be cloned using standard biobrick methods. Two single stranded, synthetic oligos were annealed to produce a double stranded DNA sequence with single stranded overhangs identical to the product of digestion by EcoRI and SpeI. Thus compatible with biobrick cloning methods.
false
IC 2010 Team
component2098249
1
BBa_K316013
component2098246
1
BBa_K316002
annotation2098249
1
BBa_K316013
range2098249
1
37
44
annotation2098246
1
BBa_K316002
range2098246
1
1
28
BBa_K316013
1
PmeI site
8bp recognition sequence for PmeI restriction endonuclease
2010-10-22T11:00:00Z
2015-05-08T01:11:56Z
This is a planning part
Information about PmeI restriction endonuclease is available at [http://www.neb.com/nebecomm/products/productR0560.asp]. The recognition site is a 8bp sequence GTTTAAAC. Pme produces a blunt cut after GTTT.
false
false
_440_
0
7480
9
Not in stock
false
This is a planning part
false
IC 2010 Team
annotation2098160
1
5' product
range2098160
1
1
4
annotation2098166
1
3' product
range2098166
1
5
8
BBa_K316002
1
Bs dif
dif excision site from B. subtilis
2010-10-19T11:00:00Z
2015-05-08T01:11:56Z
The dif sites were made by annealing synthestised oligoes.
Dif sites are naturally found in B.subtilis and are used by this organism during genome replication.
false
false
_440_
0
7480
9
Not in stock
false
The dif site was made by oligos designed to make overhangs for EcoRI and SpeI ( and ) or XbaI and PstI ( and ) to be used in standard Biobrick or 3A cloning.
false
IC 2010 Team
BBa_K143021
1
RBS-spoVG
SpoVG ribosome binding site (RBS) for B. subtilis
2008-09-16T11:00:00Z
2015-05-08T01:10:23Z
The sequence was taken from a previous research paper [1] and was constructed by Geneart.
Released HQ 2013
Description: SpoVG is an endogenous ribosome binding site from B.subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B.subtilis. Previous research showed that the predicted binding energy of the 16s rRNA to the RBS is -19kcal <cite>1</cite>
false
true
_199_
0
2090
9
In stock
false
In order to ensure that the RBS is functional the actual ribosome binding site was maintained and the distance between the RBS and the start codon maintained. In order to conform to the biobrick standard the sequence flanking the RBS had to be changed but the distance between the promoter and RBS, and start codon and RBS was maintained.
false
James Chappell
annotation1975997
1
rbs
range1975997
1
1
12
BBa_K143053
1
Pveg-spoVG
Promoter Pveg and RBS spoVG for B. subtilis
2008-10-07T11:00:00Z
2015-05-08T01:10:24Z
Pveg-spoVG was synthesised by GeneArt
Released HQ 2013
Constitutive promoter veg(<bbpart>BBa_K143012</bbpart>) coupled to the strong Ribosome Binding Site spoVG(<bbpart>BBa_K143021</bbpart>) from ''B. subtilis''.
Pveg-spoVG can be used in the context of a '''Ribosomes per second''' (RiPS) output generator
'''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix<cite>1</cite>. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
false
true
_199_
0
3475
9
In stock
false
The sequence of Pveg was obtained from the DBTBS<cite>1</cite> and RBS-spoVG were obtained from papers<cite>2</cite> and the sequence synthesised by GeneArt
true
Chris Hirst
component1979395
1
BBa_K143012
component1979397
1
BBa_K143021
annotation1979395
1
BBa_K143012
range1979395
1
1
97
annotation1979397
1
BBa_K143021
range1979397
1
106
117
BBa_K143031
1
Aad9
Aad9 Spectinomycin Resistance Gene
2008-09-15T11:00:00Z
2015-05-08T01:10:24Z
Aad9 was PCR cloned from the ''B. subtilis'' integration vector pDR111 using Pfu DNA polymerase
Aad9 is the spectinomycin resistance gene from ''Enterococcus faecalis''<cite>#1</cite>. Expression in a host confers resistance to spectinomycin at a concentration of 100μg/μl and has been used in a variety of vectors for both ''B. subtilis'' and ''E. coli'' including pDP870<cite>#2</cite>, pCOMT-Kan<cite>#3</cite> and pIEF16s<cite>#4</cite>
====References====
<biblio>
#1 pmid=1659306
#2 pmid=16997985
#3 pmid=15060042
#4 pmid=16714443
</biblio>
false
false
_199_
0
3475
9
It's complicated
false
The sequence of ''B. subtilis'' integration vector pDR111 was searched for the spectinomycin resistance gene and the Biobrick standard applied to the gene sequence upon PCR cloning
false
Chris Hirst
annotation1992697
1
stop
range1992697
1
769
771
annotation1975961
1
Aad9 Spectinomycin Acetyltransferase
range1975961
1
1
765
annotation1992698
1
start
range1992698
1
1
3
annotation1992696
1
stop
range1992696
1
766
768
BBa_K143065_sequence
1
atgaggaggatatatttgaatacatacgaacaaattaataaagtgaaaaaaatacttcggaaacatttaaaaaataaccttattggtacttacatgtttggatcaggagttgagagtggactaaaaccaaatagtgatcttgactttttagtcgtcgtatctgaaccattgacagatcaaagtaaagaaatacttatacaaaaaattagacctatttcaaaaaaaataggagataaaagcaacttacgatatattgaattaacaattattattcagcaagaaatggtaccgtggaatcatcctcccaaacaagaatttatttatggagaatggttacaagagctttatgaacaaggatacattcctcagaaggaattaaattcagatttaaccataatgctttaccaagcaaaacgaaaaaataaaagaatatacggaaattatgacttagaggaattactacctgatattccattttctgatgtgagaagagccattatggattcgtcagaggaattaatagataattatcaggatgatgaaaccaactctatattaactttatgccgtatgattttaactatggacacgggtaaaatcataccaaaagatattgcgggaaatgcagtggctgaatcttctccattagaacatagggagagaattttgttagcagttcgtagttatcttggagagaatattgaatggactaatgaaaatgtaaatttaactataaactatttaaataacagattaaaaaaattataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
BBa_K316013_sequence
1
gaaatttc
BBa_K316014_sequence
1
atctcctagaatatatattatgtaaacttactagaggaaatttc
BBa_K143009_sequence
1
gatgtacccgtttatgtgaagctatccccgaacgtggctaatatcacagaaattgcattagcgatcgaggaagcgggagcggacggtcttacgatgatcaacacactaatcggcatgagactcgatttaaaaaccggcaaaccgatattagcgaataaaacagggggactttcgggccctgctgtgaagccggttgccattcgcatggtgtatgaagtcagccagatggtcaacatcccgattatcggaatgggaggcgtgcaaacggctgaagatgccctggaatttcttctcgcgggagcaagcgcagtcgctgtcggaacagcaaactttgtgaatccttttgcatgtccagagattattgaacagctcccatctgttttgctccaatacggctatcaatcaattgaagaatgcatcggaaggagctggaatcatgaaaaacaacctgcccatcatcgcgcttg
BBa_K316002_sequence
1
atctcctagaatatatattatgtaaact
BBa_K316020_sequence
1
atgctagaggtgaaattgccgggacttgatttgaaaaacccaatcattcctgcatcaggctgcttcggttttggaaaagaattttcacgtttttatgatttgtcttgtcttggagctatcatgattaaggctacgacaaaggagccgcgctttgggaatccgacgccgcgggtagctgagactggtgctggaatgctcaatgcgatcggtctccaaaatccggggctggatagtgtgttgcatcatgagctgccgtggcttgagcagtttgatacaccgatcattgccaatgtcgcaggttctcaagtcgatgattatgttgaagtcgcagaacatatcagcaaagcgcctaatgttcatgctcttgaattgaatatttcctgcccgaatgtgaaaacaggcggaatcgcttttggcacgaatcctgaaatggctgccgatttgacaaaagcggtgaaagaggtttcgtactagagatctcctagaatatatattatgtaaacttactagagaattttgtcaaaataattttattgacaacgtcttattaacgttgatataatttaaattttatttgacaaaaatgggctcgtgttgtacaataaatgttactagagaaaggtggtgaatactagatgaggaggatatatttgaatacatacgaacaaattaataaagtgaaaaaaatacttcggaaacatttaaaaaataaccttattggtacttacatgtttggatcaggagttgagagtggactaaaaccaaatagtgatcttgactttttagtcgtcgtatctgaaccattgacagatcaaagtaaagaaatacttatacaaaaaattagacctatttcaaaaaaaataggagataaaagcaacttacgatatattgaattaacaattattattcagcaagaaatggtaccgtggaatcatcctcccaaacaagaatttatttatggagaatggttacaagagctttatgaacaaggatacattcctcagaaggaattaaattcagatttaaccataatgctttaccaagcaaaacgaaaaaataaaagaatatacggaaattatgacttagaggaattactacctgatattccattttctgatgtgagaagagccattatggattcgtcagaggaattaatagataattatcaggatgatgaaaccaactctatattaactttatgccgtatgattttaactatggacacgggtaaaatcataccaaaagatattgcgggaaatgcagtggctgaatcttctccattagaacatagggagagaattttgttagcagttcgtagttatcttggagagaatattgaatggactaatgaaaatgtaaatttaactataaactatttaaataacagattaaaaaaattataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatatactagagatctcctagaatatatattatgtaaacttactagaggaaatttctactagaggatgtacccgtttatgtgaagctatccccgaacgtggctaatatcacagaaattgcattagcgatcgaggaagcgggagcggacggtcttacgatgatcaacacactaatcggcatgagactcgatttaaaaaccggcaaaccgatattagcgaataaaacagggggactttcgggccctgctgtgaagccggttgccattcgcatggtgtatgaagtcagccagatggtcaacatcccgattatcggaatgggaggcgtgcaaacggctgaagatgccctggaatttcttctcgcgggagcaagcgcagtcgctgtcggaacagcaaactttgtgaatccttttgcatgtccagagattattgaacagctcccatctgttttgctccaatacggctatcaatcaattgaagaatgcatcggaaggagctggaatcatgaaaaacaacctgcccatcatcgcgcttg
BBa_K143008_sequence
1
atgctagaggtgaaattgccgggacttgatttgaaaaacccaatcattcctgcatcaggctgcttcggttttggaaaagaattttcacgtttttatgatttgtcttgtcttggagctatcatgattaaggctacgacaaaggagccgcgctttgggaatccgacgccgcgggtagctgagactggtgctggaatgctcaatgcgatcggtctccaaaatccggggctggatagtgtgttgcatcatgagctgccgtggcttgagcagtttgatacaccgatcattgccaatgtcgcaggttctcaagtcgatgattatgttgaagtcgcagaacatatcagcaaagcgcctaatgttcatgctcttgaattgaatatttcctgcccgaatgtgaaaacaggcggaatcgcttttggcacgaatcctgaaatggctgccgatttgacaaaagcggtgaaagaggtttcg
BBa_K143012_sequence
1
aattttgtcaaaataattttattgacaacgtcttattaacgttgatataatttaaattttatttgacaaaaatgggctcgtgttgtacaataaatgt
BBa_K143053_sequence
1
aattttgtcaaaataattttattgacaacgtcttattaacgttgatataatttaaattttatttgacaaaaatgggctcgtgttgtacaataaatgttactagagaaaggtggtgaa
BBa_B0010_sequence
1
ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc
BBa_K143021_sequence
1
aaaggtggtgaa
BBa_K143031_sequence
1
atgaggaggatatatttgaatacatacgaacaaattaataaagtgaaaaaaatacttcggaaacatttaaaaaataaccttattggtacttacatgtttggatcaggagttgagagtggactaaaaccaaatagtgatcttgactttttagtcgtcgtatctgaaccattgacagatcaaagtaaagaaatacttatacaaaaaattagacctatttcaaaaaaaataggagataaaagcaacttacgatatattgaattaacaattattattcagcaagaaatggtaccgtggaatcatcctcccaaacaagaatttatttatggagaatggttacaagagctttatgaacaaggatacattcctcagaaggaattaaattcagatttaaccataatgctttaccaagcaaaacgaaaaaataaaagaatatacggaaattatgacttagaggaattactacctgatattccattttctgatgtgagaagagccattatggattcgtcagaggaattaatagataattatcaggatgatgaaaccaactctatattaactttatgccgtatgattttaactatggacacgggtaaaatcataccaaaagatattgcgggaaatgcagtggctgaatcttctccattagaacatagggagagaattttgttagcagttcgtagttatcttggagagaatattgaatggactaatgaaaatgtaaatttaactataaactatttaaataacagattaaaaaaattataataa
BBa_B0012_sequence
1
tcacactggctcaccttcgggtgggcctttctgcgtttata
igem2sbol
1
iGEM to SBOL conversion
Conversion of the iGEM parts registry to SBOL2.1
James Alastair McLaughlin
Chris J. Myers
2017-03-06T15:00:00.000Z