This part confers resistance to Geneticin (G418) in yeast. Selection of yeast cells is typically preformed on plates with 200??g/ml of G418.
The part was synthesized from the PMKV005 plasmid used in McCusker et. al. The coding region contains the KanR open reading from transposon Tn903 from E. coli. 1. McCusker JH, Vorachek-Warren MK. DsdA (D-serine deaminase): a new heterologous MX cassette for gene disruption and selection in Saccharomyces cerevisiae. Yeast (Chichester, England). 2004;21(2):163-71. Available at: http://www.ncbi.nlm.nih.gov/pubmed/14755641.
bleR cassette, selection marker for Aspergillus nidulans
bleR cassette, antibiotic selection marker for Aspergillus nidulans
Compatible with the Plug'n'Play assembly standard
aadA Cassette + Flanked by TLTerminators (missing RBS)
KanR gene BBa_P1003 with BsmBI removed, it has duplicated promoter region for kanR since it was amplified from pSB3K5m which is a pSB3K5 derivative. Flanked with bidirectional translational Terminators BBa_J133422 (prefix) and BBa_B0042 (postfix)
BBa K125520 Aminoglycoside 3"-Adenylyltransferase (aadA1) Gene CDS
R.reniformis luciferase + ADH1 terminator + Kanamycin resistance
Zeocoin and bleomycin resistance cassette including promoter and coding sequence. It lacks a terminator.
Codon optimized for E coli by GenScript.
Streptoalloteichus hindustanus, PDB: 1BYL
Aad9 is the spectinomycin resistance gene from ''Enterococcus faecalis''#1. 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#2, pCOMT-Kan#3 and pIEF16s#4 ====References==== <biblio> #1 pmid=1659306 #2 pmid=16997985 #3 pmid=15060042 #4 pmid=16714443 </biblio>
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
Aad9 was PCR cloned from the ''B. subtilis'' integration vector pDR111 using Pfu DNA polymerase
Neomycin resistance gene. This gene can be used for selection of cells with an efficient transformation. This gene codes neomycin phosphotransferase which breaks neomycin, kanamycin, and gentamicin molecules. If you use these gene as selectivity gene in bacteria, and your laboratory lacks neomycin, you can replace it for kanamycin by adding 35 mg/ml per liter of LB media. The best concentration of neomycin to use is 80 mg/ml per liter of LB media. This gene can be also used as a selective gene for mammalian cells using gentamicin.
This gene was obtained from pCDNA 3.1 myc His(A).
aadD - Kanamycin Resistance Gene Coding Sequence
QnrS1 resistance cassette BioBrick BBa_K1930004.
To design a QnrS1 resistance cassette BioBrick BBa_K1930004 we designed a gBlock that contains the Bacillus subtilis promoter PAtpI, which is active from a very early stage of germination and includes a ribosome binding site. The gBlock also contains the original QnrS1 gene sequence from E. coli, the double terminator BBa_B0015 from iGEM as well as the prefix and suffix for BioBricks. In summary the ciprofloxacin cassette consists of the following parts PAtpI+RBS+QnrS1+2TER.
Ordered as gBlock. IDT (Integrated DNA technologies).
Origin for Asaia and resistance to Kanamycin
Hygromycin cassette for selection of mammalian cells. The selection cassette contains the SV40 promoter, the hygromycin resistance gene, and the SV40 poly A as terminator
Compatible with the Plu'n'Play assembly standard
From plasmid pcDNA3.1/Hygro from Invitrogen
streptmycin resisitance gene (can PCR ampicillin resistance gene primer)
streptmycin resisitance gene(aadA) This aadA gene are inserted into blaZ gene come from pI258 plasmid. So this aadA gene can PCR with primers for blaZ.
aminoglycoside resistance gene (aadA) come from R100.1 plasmid.
Yeast Optimized sfGFP-C + ADH1 terminator + CaUra3 Cassette
Gives resistance against the antibiotic zeocin for eucaryotes.
tetracycline resistance protein TetA(C) (backwards) [cf. BBa_J31007]
This is the coding region for Tetracyline resistance in the reverse orientation. If a promoter adn RBS are placed downstream and the entire region, from promoter to stop codon is flipped the cell will express tetracycline resistance.
ptrA, selection marker for Aspergillus nidulans
Spc - Spectinomycin Resistance Gene Coding Sequence
This part codes for the chloramphenicol acetyltransferase (cat) gene used in Bacillus subtilis for resistance against the antibiotic chloramphenicol. We constructed it in the freiburg standard (Assembly 25) for in-frame fusion-proteins. There is a Shine-Dalgarno Sequence (RBS) optimized for Bacillus subtilis included between the XbaI and NgoIV restriction site. You can use it both N-and C terminal fusions.
We constructed the part in the freiburg standard (Assembly 25). We included a Bacillus optimized RBS between the XbaI and NgoIV restriction site.
Plasmid: Bacillus subtilis reporter vector pAC6
Encryption key sequence in integration plasmid (BBa_K823023)
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The key sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted key sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our key sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation.
Key sequence: Created by our CryptoGErM software. http://2016.igem.org/Team:Groningen/Encoding BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023
Translational unit including aacC2 gene encoding gentamicin resistance (BBa_T9000) with strong RBS BBa_B0034
Escherichia coli R-Plasmid. Sequence from Genbank: http://www.ncbi.nlm.nih.gov/nuccore/X54723
For selection of E. coli transformants, contains promoter, ampR gene, and terminator
This sequence codes for a beta-lactamase, which degrades ampicillin. There is no RBS or other information attached, only the sequence for the enzyme plus prefix and suffix.
The primer sequences can be found on iGEM Team Braunschweig's wiki.
The part was derived from BBa_P1002 ampicillin resistance cassette. The sequence for the beta-lactamase was gained by PCR of the original BBa_P1002 with appropiate primers.
Chloramphenicol resistance gene Chloramphenicol is effective against a wide variety of Gram-positive and Gram-negative bacteria, including most anaerobic organisms.This part includes a gene which has resistance to chloramphenicol. In our project, we use this part as a marker to select colonies of E.coli which succeeded in transformation.
hygromycin cassette for Aspergillus nidulans
Macrolide-Lincosamide-Streptogramin B Resistance Marker
This mlsR gene was chosen as an additional selection marker for transformation of Gram positive chassis organisms. In the intended chassis organism of Clostridium acetobutylicum, the concentrations for selectivity were known, making this a convenient choice.
The cloning of this part was relatively straight-forward. No special design considerations were required.
This part was cloned from the plasmid pIKM1.
Spectinomycin R open-reading-frame basic part
JL1 Forward EcoRI oligo to bioBrick SpecR: gacttgaattcgcggccgcttctagagtccaagcgagctcgatatc JL2 Reverse SpeI oligo to bioBrick SpecR: ccgctactagtCAGCAACGATGTTACGCAGC
PCR JL1/JL2 on wt. pSPlam (917bp, EcoRI/SpeI, Neb2) Paste into pSB1A2-I13521 (EcoRI/SpeI, Neb2, Large 2062)
Ampicillin resistance gene Ampicillin acts as an irreversible inhibitor of the enzyme transpeptidase, which is needed by bacteria to make their cell walls. It inhibits the third and final stage of bacterial cell wall synthesis in binary fission, which ultimately leads to cell lysis. This part includes a gene which has resistance to ampicillin. In our project, we use this part as a marker to select colonies of E.coli which succeeded in transformation.
Ciprofloxacin resistance cassette in integration plasmid (BBa_K823023)
This part is a ciprofloxacin resistance cassette. It is containing a PAtpI constitutive promoter, ciprofloxacin resistance gene qnrS1 and double terminator. The promoter PAtpI ([http://parts.igem.org/Part:BBa_K1930005 ''BBa_K1930005'']) is a constitutive promoter which has its origin in Bacillus subtilis. It is responsible for the expression of atpA gene (ATP synthesis) during the first 30 minutes of the germination of B. subtilis. atpA gene is part of an operon (atpI-atpB-atpE-atpF-atpH-atpA-atpG-atpD-atpC), therefore the promoter region in front of the first protein coding gene (atpI) in this operon was chosen. Ciprofloxacin resistance gene qnrS1 is found naturally in E. coli, and other Gram-negative strains. qnr genes code for pentapeptide repeat proteins.??These proteins reduce susceptibility to quinolones by protecting the complex of DNA and DNA gyrase enzyme from the inhibitory effect of quinolones. Double terminator ([http://parts.igem.org/Part:BBa_B0015 ''BBa_B0015'']) is the most commonly used terminator. For more information see here: http://parts.igem.org/Part:BBa_B0015.
Ciprofloxacin resistance cassette: Ordered as gBlock. IDT (Integrated DNA technologies). BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023.
Resistance cassette for selection of mammalian cells. The cassette contains a SV40 promoter, the neomycin resistance gene, and the SV40 poly A as a terminator.
rLucC + ADH1 terminator + CaUra3 cassette
chloramphenicol acetyltransferase (forwards, CmF) [cf. BBa_J31004]
When a promoter and an RBS are in front of the gene, the cell will express Chloramphenicol resistance. Because it contains full biobrick ends, this part can be used to easily add chloramphenicol resistance to any part without changing plasmid vectors.
The bialaphos resistance (bar) gene confers resistance to bialaphos and phosphinothricin.
The plasmid sequence was unknown so extensive sequencing was carried out in order to extract it from its original vector, confirm BioBrick compatibility, and transfer it to pSB1C3.
pBARGPE1 from the FGSC (http://www.fgsc.net/fgn/pall.html)
aadA gene with lacIQ promoter and RBS and oriT
BBa_K125530 with an oriT postfixed, intermediate from the 2008 iGEM Hawaii team collection
This is a kanamycin resistance part without promotor. It is made from the BioBrick BBa_P1003 and is used for screenings.
we removed the promotor from the kanamycin resistance gene in BioBrick BBa_P1003
kanamycin resistance backwards (KanB) [cf. BBa_J23012 & BBa_J31003]
Codes the reverse of the Kanamycin Resistance gene. When the region is flipped, Kanamycin resistance will be expressed as long as a promoter and RBS are in front of the region.
tetracycline resistance protein TetA(C) (forward), [cf. BBa_J31006]
This part is the coding region of the TetR gene. It requires a promoter and RBS for the cell to be able to express tetracyline resistance.
kanamycin resistance forward (KanF) [cf. BBa_J23012 & BBa_J31002]
If there is a promoter and an RBS before this region, the cell will express Kanamycin resistance. As it has full biobricks, you can easily add KanR to an existing part without changing the plasmid vector,
chloramphenicol acetyltransferase (backwards, CmB) [cf. BBa_J31005]
Codes the reverse of the CmR gene. If the region has a promoter and an RBS in front and is flipped, the cell will express Chloramphenicol resistance.
aadA (streptomycin 3'-adenyltransferase)
This part codes for an aminoglycoside resistance protein that endows ''E. coli'' with resistance to the antibiotic streptomycin. This part can be used as a selection marker.
This part was codon-optimized for expression in E. coli. The following restriction enzyme sites were also removed: EcoRI, NotI, XbaI, SpeI, PstI, NheI, AvrII, ApoI, MfeI, NsiI, SbfI.
aadA was isolated from a plasmid in ''E. coli''. The original sequence was obtained from GenBank (VERSION NC_002525.1, GI:10955438).
P1003** Kan resistant gene with 2 Amber Codon
The amber mutations avoid the expression of the P1003 gene even at usual concentration that prevent the expression of kanamycin resistance, although one mutation (BBa_K914009) permits some leakage in translation for this gene. This mutation is rescued with the tRNA amber suppressor supD (BBa_K914000). The idea here is to prevent the kanamycin gene resistance to be expressed in case horizontal gene transfer.
I have need to design the primers that will introduce a mutation from a serine that was closer to the TAG codon (either 1, 2 or 3 mutations, here it was 1 and 2 mutations for both mutations), and it is necessary to have the amber codon in the middle of the coding sequence, not in the beginning, to be not interpreted as a alternative start codon for instance.
This part is made from the BBa_K914009 part from which I add a second mutation with the QuickChange Site-directed mutagenesis kit, Agilent.
Encrypted message sequence in integration plasmid (BBa_K823023)
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The message sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted message sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our message sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation.
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The message sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted message sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our message sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation. For more information see plasmid construction under our lab journal: http://2016.igem.org/Team:Groningen/Experiments
Message sequence: Created by our CryptoGErM software. http://2016.igem.org/Team:Groningen/Encoding BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023
Yeast Optimized sfGFP-N + ADH1 terminator + Kanamycin resistance
This is the cassette (promoter+coding region+terminator) that imparts resistance to nourseothricin in yeast.
rLucN + ADH1 terminator + Kanamycin resistance
The single mutation avoid the expression of the P1003 gene, that prevent the expression of a kanamycin resistance. This mutation is rescue with the tRNA amber suppressor supD (BBa_K914000), or any genotype relative to sup44 . The idea here is to prevent the kanamycin gene resistance in case horizontal gene transfer. Actually it would be better with another mutation.
I made the single point mutation with a quick change directed mutagenesis kit.
E.coli R-plasmid aacC2 gene for aminoglycoside-(3)-N-acetyltransferase activity AKA Gentamicin resistance
R.reniformis luciferase + ADH1 terminator + CaUra3
This part contains: - a mutated ''virG'' response regulator from the virA/G receptor system from ''Agrobacterium tumefaciens'' which works in ''Escherichia coli'' without an ''rpoA'' gene from ''A. tumefaciens'' - a kanamycin resistance under the control of a ''vir'' promotor This device is used to screen ''virA'' receptor mutants. The ''virA'' receptor is mutated in the BBa_K389010 BioBrick. The better the receptor detects a substance, the more ''virG'' is phosphorylated and the stronger the kanamycin resistance from this BioBrick is expressed. The BioBricks BBa_K389010 and BBa_K389011 have to be used together in one cell.
medium strong constitutive promotor, terminator to keep basal expression of kanamycin resistance low
synthetic ''virG'' gene, ''vir'' promotor from ''A. tumefaciens'' C58, partsregistry