Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V001284 | pFC332 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
AMA1 is a key autonomous plasmid replication element identified in Aspergillus nidulans, structurally consisting of two inverted MATE elements flanking a 0.3-kb central spacer and featuring symmetric Sau3A restriction sites at its ends. It functions as a highly efficient plasmid replicator, boosting transformation frequencies by 100- to 2000-fold and maintaining 10-20 plasmid copies per nucleus, with stable phenotypes (40-60% of spores carrying the plasmid on selective media) and minimal rearrangement or chromosomal integration. Unlike transformation enhancers like ANS1 or single MATE elements that cause plasmid rearrangement, AMA1 supports stable extrachromosomal replication independently, making it valuable for gene cloning (e.g., "instant gene bank" methods) and gene expression studies across Aspergillus, Penicillium, and other fungal genera. This is an AMA1 plasmid with Aspergillus optimized Cas9 and Hyg selection marker.
- Vector Name:
- pFC332
- Antibiotic Resistance:
- Ampicillin
- Length:
- 15559 bp
- Type:
- Bacterial Expression, CRISPR ; Fungal expression
- Replication origin:
- ori
- Selection Marker:
- Hygromycin
- Promoter:
- Aspergillus nidulans trpC promoter
- 5' Primer:
- GCTAGTGGAGGTCAACACATCAATGC
- Growth Strain(s):
- stbl3
pFC332 vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi.PMID: 26177455 PMCID: PMC4503723 DOI: 10.1371/journal.pone.0133085
pFC332 vector Sequence
LOCUS Exported 15559 bp DNA circular SYN 12-NOV-2025
DEFINITION AMA1 plasmid with Aspergillus optimized Cas9 and hph selection
marker.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15559)
AUTHORS Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH
TITLE A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi.
JOURNAL PLoS One. 2015 Jul 15;10(7):e0133085. doi:
10.1371/journal.pone.0133085. eCollection 2015.
PUBMED 26177455
REFERENCE 2 (bases 1 to 15559)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 15559)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 15559)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 15559)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
date: "2015-07-15"; pages: "
10.1371/journal.pone.0133085. eCollection 2015"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..15559
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(2..859)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(860..951)
/gene="bla"
/label=AmpR promoter
primer_bind 993..1012
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 1018..1037
/label=pENTR-R
/note="pENTR vectors, reverse primer"
protein_bind 1244..1277
/label=FRT (minimal)
/note="supports FLP-mediated excision but not integration
(Turan and Bode, 2011)"
rep_origin 1605..6822
/label=AMA1
/note="replication origin from Aspergillus nidulans
(Aleksenko and Clutterbuck, 1996)"
promoter 7073..8008
/label=pTEF1
/note="Aspergillus nidulans TEF promoter"
CDS 8009..12112
/codon_start=1
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 12116..12136
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
terminator 12140..12628
/label=TEF1-TT
/note="Aspergillus nidulans TEF Terminator; TEF1-TT"
promoter 12638..12961
/label=PtrpC
/note="tryptophan synthase promoter (PtrpC)"
CDS 12962..13984
/codon_start=1
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANILFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
protein_bind complement(14059..14092)
/label=FRT (minimal)
/note="supports FLP-mediated excision but not integration
(Turan and Bode, 2011)"
primer_bind complement(14562..14579)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(14677..14694)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(14848..15436)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"