Parts used
| Code |
Name |
Type |
Part |
Description |
| BBa_K5316002 |
EF1A promoter |
Promoter |
Newly registered |
Human eukaryotic translation elongation factor 1 α1 (EF1A) promoter is a strong, constitutive, and ubiquitous mammalian promoter. |
| BBa_K5316019 |
CMV promoter |
Promoter |
Newly registered |
The human cytomegalovirus immediate early (CMV) promoter is a strong, constitutive, and ubiquitous promoter. |
| BBa_K4046050 |
Kozak sequence |
Regulatory |
Registered |
Facilitates translation initiation of ATG start codon downstream of the Kozak sequence. |
| BBa_K5316006 |
hBDNF |
CDS |
Newly registered |
Brain-Derived Neurotrophic Factor (BDNF) is a protein that plays a key role in supporting the growth, survival, and differentiation of neurons. |
| BBa_K5316007 |
H2B |
Signal Peptide |
Newly registered |
Histone H2B is a core component of the nucleosome, commonly fused to fluorescent proteins to guide these proteins to the nucleus. |
| BBa_K4839013 |
mCherry |
CDS |
Registered |
mCherry is a red fluorescent protein that was developed as a variant of mRFP1 through mutagenesis. |
| BBa_K4251016 |
BGH pA |
PolyA |
Registered |
The bovine growth hormone polyadenylation signal (BGH pA) is a widely used sequence that facilitates proper transcription termination and polyadenylation of mRNA. |
| BBa_K5316008 |
5’ ITR |
Packaging |
Newly registered |
The adeno-associated virus (AAV) 5' inverted terminal repeat (ITR) is a key regulatory sequence that plays a crucial role in enabling the rescue of the viral genome from recombinant plasmids. |
| BBa_K1680005 |
LoxP |
Recognition |
Registered |
The loxP site (Locus of X-over in P1) is a specific DNA sequence that serves as the recognition site for Cre recombinase, an enzyme derived from the P1 bacteriophage. |
| BBa_K4814003 |
3xGGGGS |
Linker |
Registered |
The 3xGGGGS linker is a commonly used flexible peptide linker that facilitates the fusion of two proteins while maintaining their individual functionality. |
| BBa_K5316010 |
ORF Stuffer |
DNA |
Newly registered |
The ORF Stuffer, composed of amino acids 2-83 of E. coli β-galactosidase, functions as a negative open reading frame (ORF) control. |
| BBa_K5316011 |
SV40 late pA |
PolyA |
Newly registered |
The SV40 late polyadenylation signal (SV40 late pA) is derived from the simian virus 40. It facilitates transcription termination and polyadenylation of mRNA. |
| BBa_K5316012 |
bfVFP |
CDS |
Newly registered |
Branchiostoma floridae Violet Fluorescent Protein (bfVFP) is a photostable and pH-insensitive protein with an emission wavelength of 430 nm. |
| BBa_K1071009 |
EGFP |
CDS |
Registered |
|
| BBa_K5316013 |
SVFP |
CDS |
Newly registered |
Sirius Violet Fluorescent Protein is a pH-insensitive and photostable protein with an emission wavelength of 424 nm. |
| BBa_K5316014 |
COX8 signal |
Signal Peptide |
Newly registered |
The COX8 presequence is a mitochondrial signal peptide derived from cytochrome c oxidase subunit VIII. It is commonly used to direct fusion proteins to the mitochondria. |
| BBa_K5316015 |
Lck signal |
Signal Peptide |
Newly registered |
The Lck signal peptide is a membrane-targeting sequence derived from the lymphocyte-specific protein tyrosine kinase. |
| BBa_K5316016 |
Lysozyme signal |
Signal Peptide |
Newly registered |
The lysozyme signal sequence is a short peptide that directs fusion proteins to the endoplasmic reticulum. |
| BBa_K5316017 |
Ampicillin resistance (BsaI clean) |
CDS |
Newly registered |
This new variant of the gene has been modified to be completely compatible with the Type IIS assembly system. |
| BBa_K5316020 |
H2B-mCherry |
CDS |
Newly registered |
Composite part |
Part Collection
Our work led us to realize that the use of reporters targeted to specific cellular locations could be highly helpful to other iGEM teams.
We therefore generated a library of different Parts for commonly-used Signal Peptides (SP) and Fluorescent Proteins (FP), including the overhangs specifically adapted to fit the designated positions in the backbone, assuring compatibility.
Since our backbone has been designed to be compatible with the iGEM kit, we have been able to use Parts from previous iGEM teams, so we have included them in the library. Additionally, to contribute more to the community, we have also added three new SP and two FP. This will allow teams to target new cellular locations with their reporters and to use new colors.
It’s important to emphasize that all overhangs in this design can be easily added to other reporters, making them compatible with the cassette. This means that other teams can incorporate any fluorescent protein or signal peptide of their choice, even if they aren’t already included in this library.
Visit our FP Handbook to find more information on how to use any piece with our backbone.
Library
Fluorescent Proteins
| Name |
Registry Code |
TIIS RE |
Flanking Compatibility |
Status |
| eCFP |
BBa_E0020 |
BsaI |
AATG/GCTT |
Existing part |
| eYFP |
BBa_E0030 |
BsaI |
AATG/GCTT |
Existing part |
| TnRFP |
BBa_J97003 |
BsaI |
AATG/GCTT |
Existing part |
| EGFP |
BBa_K1071009 |
BsaI |
AATG/GCTT |
Existing part |
| bfVFP |
BBa_K5316012 |
BsaI |
AATG/GCTT |
New part |
| SVFP |
BBa_K1071009 |
BsaI |
AATG/GCTT |
New part |
Signal Peptides
| Localization |
Part Name |
TIIS RE |
Flanking Compatibility |
Status |
| Membrane |
BBa_K5316015 |
Esp3I |
ACGA/TCTC |
New Part |
| Mitochondria |
BBa_K5316014 |
Esp3I |
ACGA/TCTC |
New Part |
| Nucleous |
BBa_K5316014 |
Esp3I |
ACGA/TCTC |
New Part |
| Endoplasmic Reticulum |
BBa_K5316016 |
Esp3I |
ACGA/TCTC |
New Part |
Customizable Transfection Reporter Cassette sequence
We have made the full sequence of the backbone available for everyone. To work with it, copy the following annotated sequence:
LOCUS VB240902-1538qea 3705 bp DNA circular SYN 03-SEP-2024
COMMENT Vector Name: pRP[Exp]-BbsI/CMV>Esp3I/{H2B}/Esp3I/3xGGGGS/BsaI/ORF_Stuffer/BsaI:SV40 pA/BbsI
COMMENT Vector Type: Mammalian Gene Expression Vector
COMMENT Plasmid Copy Number: High
COMMENT Antibiotic Resistance: Ampicillin
FEATURES Location/Qualifiers
Miscellaneous 1..6
/label="BsiWI"
/Description="BsiWI restriction site"
/Application Notes="Allows linearization of vector."
/Full Name="BsiWI restriction enzyme site"
/color="#800C0C"
Miscellaneous 7..14
/label="AscI"
/Description="AscI restriction site"
/Application Notes="Allows linearization of vector."
/Full Name="AscI restriction enzyme site"
/color="#0C5D90"
misc_feature 46..51
/label="BbsI"
/color="#F58600"
misc_feature 52..640
/label="CMV"
/Application Notes="Strong promoter; may have variable strength in some cell types."
/Full Name="Human cytomegalovirus immediate early promoter"
/Subtype="Mammalian ubiquitous promoter"
/References="Cell. 41:521 (1985); PLoS One. 5:e10611 (2010)"
/color="#5566F5"
misc_feature 670..675
/label="Esp3I"
/color="#EF6CDF"
misc_feature 676..681
/label="Kozak"
/Application Notes="Facilitates translation initiation of ATG start codon downstream of the Kozak sequence."
/Full Name="Kozak"
/References="Nucleic Acids Res. 9:5233 (1981)"
/color="#C54B7C"
misc_feature 682..1059
/label="H2B"
/Subtype="Fluorescent reporter"
/References="Nat Biotechnol. 22:1567 (2004)"
/color="#D05C0A"
misc_feature 1062..1067
/label="Esp3I"
/Full Name="detaBbsI restriction enzyme site"
/color="#FD3434"
misc_feature 1075..1122
/label="3xGGGGS"
/Application Notes="Commonly used flexible linker for fusing two proteins."
/Full Name="3 tandem GGGGS"
/Subtype="Read-through linker"
/References="Adv Drug Deliv Rev. 65: 1357 (2013)"
/color="#46C6EF"
misc_feature 1130..1135
/label="BsaI"
/color="#EDA481"
CDS 1136..1384
/label="ORF_Stuffer"
/Description="Amino acid 2-83 of E. coli beta-galactosidase"
/Application Notes="Can be used as a negative ORF control."
/Subtype="Stuffer"
/References="Designed by VectorBuilder"
/color="#DA3BB5"
misc_feature 1385..1390
/label="BsaI"
/Full Name="BsaI restriction enzyme site"
/color="#E4B930"
polyA_signal 1440..1661
/label="SV40 late pA"
/Description="Simian virus 40 late polyadenylation signal"
/Application Notes="Allows transcription termination and polyadenylation of mRNA transcribed by Pol II RNA polymerase."
/References="Mol. Cell. Biol.9:4248 (1989)"
/color="#7A00B5"
misc_feature 1662..1667
/label="BbsI"
/Full Name="detaBbsI restriction enzyme site"
/color="#05696D"
Miscellaneous 1691..1698
/label="PacI"
/Description="PacI restriction site"
/Application Notes="Allows linearization of vector."
/Full Name="PacI restriction enzyme site"
/color="#F54C6B"
Rep_origin complement(1886..2474)
/label="pUC ori"
/Description="pUC origin of replication"
/Application Notes="Facilitates plasmid replication in E. coli; regulates high-copy plasmid number (500-700)."
/Full Name="pUC origin of replication"
/Subtype="E. coli replication_origin"
/References="Mol Microbiol. 6:3385 (1992)"
/color="#944603"
CDS complement(2645..3505)
/label="Ampicillin"
/Description="AmpiciIIin resistance gene"
/Application Notes="Allows E. coli to be resistant to ampicillin."
/Selection Drug="Ampicillin "
/Usage="Positive selection"
/Full Name="Ampicillin resistance gene"
/Subtype="E. coli selection marker"
/References="Proc Natl Acad Sci U S A. 75: 3737 (1978)"
/color="#CE0084"
ORIGIN
1 CGTACGGGCG CGCCGCGGCC GCCAACTTTG TATAGAAAAG TTGCAGTCTT CTAGTTATTA
61 ATAGTAATCA ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA
121 ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT TGACGTCAAT
181 AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA
241 GTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTACGCC
301 CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT
361 ATGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGTGAT
421 GCGGTTTTGG CAGTACATCA ATGGGCGTGG ATAGCGGTTT GACTCACGGG GATTTCCAAG
481 TCTCCACCCC ATTGACGTCA ATGGGAGTTT GTTTTGGCAC CAAAATCAAC GGGACTTTCC
541 AAAATGTCGT AACAACTCCG CCCCATTGAC GCAAATGGGC GGTAGGCGTG TACGGTGGGA
601 GGTCTATATA AGCAGAGCTG GTTTAGTGAA CCGTCAGATC CAAGTTTGTA CAAAAAAGCA
661 GGCTACGATG AGACGGCCAC CATGCCAGAG CCAGCGAAGT CTGCTCCCGC CCCGAAAAAG
721 GGCTCCAAGA AGGCGGTGAC TAAGGCGCAG AAGAAAGGCG GCAAGAAGCG CAAGCGCAGC
781 CGCAAGGAGA GCTATTCCAT CTATGTGTAC AAGGTTCTGA AGCAGGTCCA CCCTGACACC
841 GGCATTTCGT CCAAGGCCAT GGGCATCATG AATTCGTTTG TGAACGACAT TTTCGAGCGC
901 ATCGCAGGTG AGGCTTCCCG CCTGGCGCAT TACAACAAGC GCTCGACCAT CACCTCCAGG
961 GAGATCCAGA CGGCCGTGCG CCTGCTGCTG CCTGGGGAGT TGGCCAAGCA CGCCGTGTCC
1021 GAGGGTACTA AGGCCATCAC CAAGTACACC AGCGCTAAGG GCGTCTCATC TCTTTCTGGT
1081 GGCGGAGGCT CGGGCGGAGG TGGGTCGGGT GGCGGCGGAT CAGGAATGCG AGACCGTCGT
1141 TTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC TTGCAGCACA
1201 TCCCCCTTTC GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA
1261 GTTGCGCAGC CTGAACGGCG AGTGGCGCTT TGCCTGGTTT CCGGCACCAG AAGCGGTGCC
1321 GGAAAGCTGG CTGGAGTGCG ATCTTCCTGA GGCCGATACT GTCGTCGTCC CCTCAAACTG
1381 GCAGGGTCTC AGCTTACCCA GCTTTCTTGT ACAAAGTGGT GATGGCCGGC CGCTTCGAGC
1441 AGACATGATA AGATACATTG ATGAGTTTGG ACAAACCACA ACTAGAATGC AGTGAAAAAA
1501 ATGCTTTATT TGTGAAATTT GTGATGCTAT TGCTTTATTT GTAACCATTA TAAGCTGCAA
1561 TAAACAAGTT AACAACAACA ATTGCATTCA TTTTATGTTT CAGGTTCAGG GGGAGGTGTG
1621 GGAGGTTTTT TAAAGCAAGT AAAACCTCTA CAAATGTGGT AGAAGACACC AACTTTATTA
1681 TACATAGTTG TTAATTAAGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG
1741 TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG
1801 AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC
1861 GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA
1921 AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT
1981 TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC
2041 TGTCCGCCTT TCTCTCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC
2101 TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC
2161 CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT
2221 TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG
2281 CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGAACA GTATTTGGTA
2341 TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA
2401 AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA
2461 AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG
2521 AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC ACCTAGATCC
2581 TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA ACTTGGTCTG
2641 ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTTCAT
2701 CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG
2761 GCCCCAGTGC TGCAATGATA CCGCGAGATC CACGCTCACC GGCTCCAGAT TTATCAGCAA
2821 TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA TCCGCCTCCA
2881 TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT AATAGTTTGC
2941 GCAACGTTGT TGCCATTGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT GGTATGGCTT
3001 CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAA
3061 AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT
3121 CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC GTAAGATGCT
3181 TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG CGGCGACCGA
3241 GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA ACTTTAAAAG
3301 TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA CCGCTGTTGA
3361 GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTTTCA
3421 CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG GGAATAAGGG
3481 CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA AGCATTTATC
3541 AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAATAG
3601 GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT CTAAGAAACC ATTATTATCA
3661 TGACATTAAC CTATAAAAAT AGGCGTATCA CGAGGCCCTT TCGTC
//