LOCUS       sequence-292473-        7479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recyclable marker for use in Cryptococcus neoformans. Contains the 
            amdS2 counterselectable marker flanked by the mRuby3 ORF and a 
            flexible linker at the 5' end..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7479)
  AUTHORS   Spencer GWK, Chua SMH, Erpf PE, Wizrah MSI, Dyba TG, Condon ND, 
            Fraser JA
  TITLE     Broadening the spectrum of fluorescent protein tools for use in the 
            encapsulated human fungal pathogen Cryptococcus neoformans.
  JOURNAL   Fungal Genet Biol. 2020 May;138:103365. doi: 
            10.1016/j.fgb.2020.103365. Epub 2020 Mar 4.
  PUBMED    32145317
REFERENCE   2  (bases 1 to 7479)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Fungal 
            Genet Biol."; date: "2020-05"; volume: "138:103365. doi"; pages: " 
            10.1016/j.fgb.2020.103365. Epub 2020 Mar 4"
COMMENT     Sequence Label: pPEE46
FEATURES             Location/Qualifiers
     source          1..7479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2169..2189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             3975..5621
                     /codon_start=1
                     /gene="amdS"
                     /product="acetamidase from Aspergillus nidulans"
                     /label=amdS
                     /note="confers ability to process acetamide for selection 
                     in fungal cells"
                     /translation="MPQSWEELAADKRARLAKTIPDEWKVQTLPAEDSVIDFPKKSGIL
                     SEAELKITEASAADLVSKLAAGELTSVEVTLAFCKRAAIAQQLTNCAHEFFPDAALAQA
                     RELDEYYAKHKRPVGPLHGLPISLKDQLRVKGYETSMGYISWLNKYDEGDSVLTTMLRK
                     AGAVFYVKTSVPQTLMVCETVNNIIGRTVNPRNKNWSCGGSSGGEGAIVGIRGGVIGVG
                     TDIGGSIRVPAAFNFLYGLRPSHGRLPYAKMANSMEGQETVHSVVGPITHSVEDLRLFT
                     KSVLGQEPWKYDSKVIPMPWRQSESDIIASKIKNGGLNIGYYNFDGNVLPHPPILRGVE
                     TTVAALAKAGHTVTPWTPYKHDFGHDLISHIYAADGSADVMRDISASGEPAIPNIKDLL
                     NPNIKAVNMNELWDTHLQKWNYQMEYLEKWREAEEKAGKELDAIIAPITPTAAVRHDQF
                     RYYGYASVINLLDFTSVVVPVTFADKNIDKKNESFKAVSELDALVQEEYDPEAYHGAPV
                     AVQVIGRRLSEERTLAIAEEVGKLLGNVVTP"
     primer_bind     complement(6793..6809)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6794..6810)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(6834..6853)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6835..6853)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6863..6880)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6863..6879)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6872..6894)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(7020..7476)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7158..7179)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7370..7389
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg
     2221 gccgctctag atggtctcta agggcgagga gctcatcaag gagaacatgc gtatgaaggt
     2281 cgtcatggag ggttctgtca acggccacca gttcaagtgc actggtgagg gtgagggcag
     2341 gccctacgag ggtgtccaga ccatgaggat caaggtcatc gagggtggtc ccctcccttt
     2401 cgccttcgac attcttgcca cctctttcat gtacggctcc cgtaccttca tcaagtaccc
     2461 cgccgacatc cctgacttct tcaagcagtc cttccctgag ggtttcactt gggagagggt
     2521 taccaggtac gaggacggtg gtgtcgtcac cgtcacccag gacacctccc ttgaggacgg
     2581 cgagctcgtc tacaacgtca aggtcagggg cgttaacttc ccctccaacg gtcccgtcat
     2641 gcagaagaag accaagggtt gggagcctaa cactgagatg atgtaccctg ctgacggtgg
     2701 tctcaggggt tacactgaca tcgctctcaa ggttgacggt ggtggcggcc gccattctat
     2761 gctatacggt acaagattgt ggctactatg caagctccat agcgaatgat cttacactga
     2821 ccttgtcgac cgttctgaat gattaacaat ggtcgcaagt gtggttgacc cacctgcggg
     2881 cgcttcttaa ctatctatca tcggtatgtc aagtatcaca ccctttaaac aaaagaataa
     2941 gagcataata cagcagtctc atcatggcaa cccttcgcgc aatgtacaaa aagcagccag
     3001 aagcccccac gcacaagcaa taatgtcgtg ttctaccctg ccatgaacaa gcgtcatggc
     3061 cattagcgaa cgccccaata ttcagcccca ttcgccaagc aagttgcctc gcccccttcc
     3121 cagaagatca tgggcatctc gtcttcttct aatttcgcat tttgtgaagc tccattcatg
     3181 cacgtcggct tctggacgac tcttatcata tgcattggcc tgctcggtgt gaggatgagc
     3241 cactcccaca tgtttagacg gcaggggcct cttgccaccc gttgaatttt tggtgcgaag
     3301 acgtgcctgg gaacgacctc cactttaaat ttctccgttg ttgcggtcgc tccatcctcc
     3361 tcacatttct tgcctcctaa gtcccatgtg ccacactcgg tgaaaaacgg agtttgatac
     3421 ggatgaaatt tccagtgagc ttttcacatt ctggaggttt cgaaaaaaat tgtgggattt
     3481 tctgaattaa tccgagaatc cgccctgccc atcaaccgga aagaattaaa tccctcgcac
     3541 attcctttct cccaccacct tctttatctc tacattcttt aaccaaaaca cctaggtgag
     3601 tacccaaagt cttttgacac ctttgaaaat ccgcgccagt cgtagggcac gtaaaacaat
     3661 tctcgctggt ggcttctgca ggtgaagtcg gggcagagtg gagctgagca tggtcgacca
     3721 cccacatcat tctgtcttgt ttttggcgat gtcttccatg cttttgccct catctgttgc
     3781 gtcgactgca gagtactgtt ttgccactac tgagctcaac atgggcttgt gtgtggtgaa
     3841 gaaaaaaatt tggtggctct tgaaaatttt caacccatcg gatacatgcc cttccaactc
     3901 acaacgccct aacactttat tgtcctatca ttgcctcttc gcacatgcta acgatctcca
     3961 cagaaaactt caaaatgcct caatcctggg aagaactggc cgctgataag cgcgcccgcc
     4021 tcgcaaaaac catccctgat gaatggaaag tccagacgct gcctgcggaa gacagcgtta
     4081 ttgatttccc aaagaaatcg gggatccttt cagaggccga actgaagatc acagaggcct
     4141 ccgctgcaga tcttgtgtcc aagctggcgg ccggagagtt gacctcggtg gaagttacgc
     4201 tagcattctg taaacgggca gcaatcgccc agcagttaac aaactgcgcc cacgagttct
     4261 tccctgacgc cgctctcgcg caggcaaggg aactcgatga atactacgca aagcacaaga
     4321 gacccgttgg tccactccat ggcctcccca tctctctcaa agaccagctt cgagtcaagg
     4381 gctacgaaac atcaatgggc tacatctcat ggctaaacaa gtacgacgaa ggggactcgg
     4441 ttctgacaac catgctccgc aaagccggtg ccgtcttcta cgtcaagacc tctgtcccgc
     4501 agaccctgat ggtctgcgag acagtcaaca acatcatcgg gcgcaccgtc aacccacgca
     4561 acaagaactg gtcgtgcggc ggcagttccg gtggtgaggg tgcgatcgtt gggattcgtg
     4621 gtggcgtcat cggtgtagga acggatatcg gtggctcgat tcgagtgccg gccgcgttca
     4681 acttcctgta cggtctaagg ccgagtcatg ggcggctgcc gtatgcaaag atggcgaaca
     4741 gcatggaggg tcaggagacg gtgcacagcg ttgtcgggcc gattacgcac tctgttgagg
     4801 acctccgcct cttcaccaaa tccgtcctcg gtcaggagcc atggaaatac gactccaagg
     4861 tcatccccat gccctggcgc cagtccgagt cggacattat tgcctccaag atcaagaacg
     4921 gcgggctcaa tatcggctac tacaacttcg acggcaatgt ccttccacac cctcctatcc
     4981 tgcgcggcgt ggaaaccacc gtcgccgcac tcgccaaagc cggtcacacc gtgaccccgt
     5041 ggacgccata caagcacgat ttcggccacg atctcatctc ccatatctac gcggctgacg
     5101 gcagcgccga cgtaatgcgc gatatcagtg catccggcga gccggcgatt ccaaatatca
     5161 aagacctact gaacccgaac atcaaagctg ttaacatgaa cgagctctgg gacacgcatc
     5221 tccagaagtg gaattaccag atggagtacc ttgagaaatg gcgggaggct gaagaaaagg
     5281 ccgggaagga actggacgcc atcatcgcgc cgattacgcc taccgctgcg gtacggcatg
     5341 accagttccg gtactatggg tatgcctctg tgatcaacct gctggatttc acgagcgtgg
     5401 ttgttccggt tacctttgcg gataagaaca tcgataagaa gaatgagagt ttcaaggcgg
     5461 ttagtgagct tgatgccctc gtgcaggaag agtatgatcc ggaggcgtac catggggcac
     5521 cggttgcagt gcaggttatc ggacggagac tcagtgaaga gaggacgttg gcgattgcag
     5581 aggaagtggg gaagttgctg ggaaatgtgg tgactccata gaaggttgag cgcgtaatag
     5641 tgggttggtc ttgtctctat ccattcatct gtgagacata ctgatttcaa ctttttcatc
     5701 agataataag gatggtgtga gataagcaag gctgggttat gaggtttggg actagatggt
     5761 gtttcgcgtc taaaggtgag tcttcgttgg ttcaatcaca ttgatctgat cgctgatcaa
     5821 ttgtcagcct ctgaatgtgc tcttgaagaa aatggttgtc tgtaagaatc cagaaccaaa
     5881 aacggtcatt tcaagtatgt atcctccgtg aagttccttt agctcttgct aatattggac
     5941 tttagcgtgc tttagccaaa gacagggtcg cgaggttaag aggatgggcc aggaaaaatc
     6001 taagttagat ttgtcacaca aagaaaaaga ttgtctcagt tttggttttg tttggtttga
     6061 tttatgcata ggaaatattt atgatcgaaa tcactcattt cctgcttcgt aaatatcatc
     6121 tactatatgc ttcgtaaatg tccatctcct acatattgtg gatatgataa atgcaatcct
     6181 ctcgtctaca ctctcggctt atgcctttgt cttctttgcg ctccttttag ccttcttttt
     6241 attcctagtc ttcaagccca tcgtacggct cccgtacctt catcaagtac cccgccgaca
     6301 tccctgactt cttcaagcag tccttccctg agggtttcac ttgggagagg gttaccaggt
     6361 acgaggacgg tggtgtcgtc accgtcaccc aggacacctc ccttgaggac ggcgagctcg
     6421 tctacaacgt caaggtcagg ggcgttaact tcccctccaa cggtcccgtc atgcagaaga
     6481 agaccaaggg ttgggagcct aacactgaga tgatgtaccc tgctgacggt ggtctcaggg
     6541 gttacactga catcgctctc aaggttgacg gtggtggcca cctccactgc aacttcgtca
     6601 ctacttacag gtctaagaag accgtcggca acatcaagat gcccggtgtc cacgccgttg
     6661 accaccgtct cgagaggatc gaggagtccg acaacgagac ctacgttgtc cagagggagg
     6721 tcgctgttgc caagtactcc aaccttggtg gtggcatgga cgagctctac aagaattcga
     6781 tatcaagctt atcgataccg tcgacctcga gggggggccc ggtacccaat tcgccctata
     6841 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     6901 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     6961 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     7021 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     7081 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     7141 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     7201 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     7261 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     7321 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     7381 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     7441 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcag
//
