Cryptococcus neoformans Database in Synthetic Biology Open Language
This is a collection of Biological Genetic Parts of Cryptococcus neoformans containing 23 different promoters, coding sequences and terminators, which were previously used and whose efficacy has already been proven in this organism. This work was made in order to facilitate the build and assembly of genetic circuits and plasmids for the study of this pathogen.
The parts were obtain through a extensive literature review and by search of plasmids in databases such as NCBI and AddGene.
Organism: Cryptococcus neoformans var. grubi H99
This is the terminator of mitogen-activated protein kinase (HOG1).
This terminator is delimited by primers present in the reference article. There is a SNP in relation to the genomic sequence (CP003830). We used the genomic sequence.
GenBank: CP003830 Region: 176667..176966 Plasmid: YSBE 508 (#92081)
Organism: Cryptococcus neoformans var. neoformans JEC21
This is the promoter of glyceraldehyde-3-phosphate dehydrogenase.
This sequence was directly sent personally by Dr. Chen Ding. We did a BLAST and found a deletion of five nucleotides in comparison with the Cryptococcus neoformans var. neoformans JEC21 chromosome sequence. Thus, we used the genomic sequence (AE017346).
GenBank: AE017346 Region: 940695..942557
Organism: Cryptococcus neoformans var. grubii H99 (Cryptococcus neoformans serotype A)
The plasmid pSDMA66 (#128355) sequence has an A in position 84, and the chromosome sequence has a G. We used the last one.
Plasmid pSDMA66 (#12835) GenBank: CP003820 Region: 1242235..12430676
Organism: Cryptococcus neoformans serotype A strain H99O
This is the acetamidase coding sequence.
amdS as a selective marker for C. neoformans.
Plasmid pPEE7 (#128357)
Specific to RNApol lII. Available on Reference Article's Supplementary Material. We couldn't find the correspondent GenBank ID because this sequence was too small.
Available on the Supplementary Material of WANG, et al. 2016.
Organism: Cryptococcus neoformans var. grubii H99
This is the promoter of the UDP-glucose epimerase.
This promoter UGE2 is repressible by glucose and inducible by galactose. The 3' primer used to amplify the UGE2 promoter region encopassed the first codons of UGE2 protein. This region was, therefore, excluded from the promoter presented here, diminishing the size of this promoter from 1007 bp to 978 bp.
GenBank: CP003831 Region: 176453..177430
Organism: Cryptococcus neoformans serotype A H99S strain
This is the coding sequence of the Nourseothricin Resistance gene (NAT/NrsR).
We deleted the restriction enzyme sequence of NotI and added the atg codon the beginning of the sequence.
Plasmid YSBE 557 (#92085)
This is the galactose transferase promoter.
This promoter, GAL7 + 5'UTR, is repressible by glucose and inducible by galactose. The 3' primer used to amplify the GAL7 promoter region encopassed the first codon ATG. This ATG codon was, therefore, excluded from the promoter presented here, diminishing the size of this promoter from 1022 to 1020 bp.
GenBank: CP003831 Region: 178967..179986
This is the terminator of the Elongation Factor 1.
TEF1 terminator has 100% identity to the Cryptococcus neoformans genomic sequence (CP003831).
GenBank: CP003831 Region: 375181..376053 Plasmid: pSDMA65 (#128354)
This is the coding sequence of the Fluorescent Protein mCherry.
The atg codon was added at the beginning of the sequence. The authors reported at the Addgene website that: “Addgene's sequencing results found K128R and Q193P mutations in mCherry. These mutations are not known to affect plasmid function”.
Plasmid YSBE 596 (#92088)
This is the coding sequence of the Neomycin Resistance gene (NEO/G418).
Plasmid pPEE33 (#128373)
Organism: Cryptococcus neoformans var. neoformans B-3501A
This is the coding sequence of the Orotidine Monophosphate Pyrophosphorylase.
This genomic sequence (CM000046) is the same sequence described in the reference article (M34606).
Genbank: CM000046 Region: 302227..303005 and M34606 Region: 714..1492
This is the coding sequence of CRISPR associated protein 9 from the widely applied CRISPR-Cas system gene editing tool.
Originally, the Cas9 nucleotide sequence was PCR amplified from the Addgene plasmid #43802, and it has 2 SNPs in relation to the original one, but both sequences code the same aminoacid. At the C- terminal end of Cas9, there is the Nuclear Localization Signal (NLS), SV40 NLS.
Plasmid pSDMA65 (#128354) Plasmid p414-TEF1p-Cas9-CYC1t (#43802)
This is the promoter of the Orotidine Monophosphate Pyrophosphorylase.
We did a BLAST with the article's sequence (M34606) and we found two deletions. Thus, the sequence provided here is the updated one, the genomic sequence (CM000046), which has 2 more bp.
GenBank: CM000046 Region: 301512..302226
This is the promoter of the Elongation Factor 1.
The plasmid TEF1 sequence (#128354) has an SNP in position 168 compared to the genomic sequence (CP003831). We used the genomic sequence.
Plasmid pSDMA65 (#128354) Genbank: CP003831 Region: 377755..378976
That is an endogenous promoter of C. neoformans serotype D strain JEC21. The sequence is available on Reference Article's Supplementary Material and it presented 100% identity with C.neoformans genomic sequence (AE017348).
GenBank: AE017348.1 Region: 871991..872263
This is the terminator of the Orotidine Monophosphate Pyrophosphorylase.
The Ura5 terminator (M34606) given by the reference has some SNPs compared to the genomic sequence (CM000046) present in this database.
GenBank: CM000046 Region: 303006..303540
Organism: Cryptococcus neoformans serotype A H99S
This is the coding sequence of the Green Fluorescent Protein (GFP).
The atg codon was added at the beginning of the sequence. Present in plasmids: YSBE 508, YSBE 557 e YSBE 608 of the same article.
Plasmid YSBE 557 (#92085) Plasmid YSBE 608 (#92089) Plasmid YSBE 508 (#92081)
This is the promoter of antiphagocytic protein.
In this article they renamed the previously isolated IP1 to antiphagocytic protein 1 (App1). The chromosome sequence (CP047908) has some SNP's and gaps compared to the article sequence (AF180108). We used the chromosome sequence.
GenBank: CP047908 Region: 1244167..1244926
This is the coding sequence of the Hygromycin Resistance gene.
Plasmid pPEE38 (#128378)
This is the galactose-1-fosfato uridililtransferase promoter.
The GAL7 promoter is induced by galactose and repressed by glucose. The 3' primer used to amplify the GAL7 promoter region encopassed part of the first exon, therefore this part was excluded from the promoter presented here, diminishing the size of this promoter from 582 to 471 bp. Some SNPs were also detected in relation to the sequence presented in the original article, therefore the genomic sequence (AE017353) was used here.
GenBank: AE017353 Region: 190704..191174
This promoter, GAL1 + 5'UTR, is repressible by glucose and inducible by galactose. The 3' primer used to amplify the GAL1 promoter region encompassed the first codon ATG. This ATG codon was, therefore, excluded from the promoter presented here, diminishing the size of this promoter from 978 to 976 bp.
Genbank: CP003831 Region: 176110..177085
Terminator sequence of Phosphoribosylanthranilate Isomerase.
We used the genomic sequence (CP003828), that has a SNP, when compared with the sequence of the plasmids presented in the reference.
Plasmid pPEE33 (#128373) Plasmid pPEE38 (#128378) GenBank: CP003828 Region: 1023919..1024166
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