LOCUS       sequence-313808-        7516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recyclable marker for use in Cryptococcus neoformans. Contains the 
            amdS2 counterselectable marker flanked by the mMaroon1 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 7516)
  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 7516)
  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: pPEE43
FEATURES             Location/Qualifiers
     source          1..7516
                     /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             3992..5638
                     /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(6830..6846)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6831..6847)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(6871..6890)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6872..6890)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6900..6917)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6900..6916)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6909..6931)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(7057..7513)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7195..7216)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7407..7426
                     /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 atggtctcca agggcgagga gctcatcaag gagaacatgc acaccaagct
     2281 ctacctcact ggcaccgtca acaaccacta cttcgagtgc accgccgagg gcgagggcaa
     2341 gccctacgag ggcacccaga ccaacaggat caaggtcgtc cgtggtggcc ccctcccctt
     2401 cgctttcgac atcctcgccc cctgcttcat gtacggctcc aagaccttca ttaaccaccc
     2461 tcctgacatt cccgactact tcaagcagtc cttccctgag ggcttcactt gggagaggac
     2521 caccgtttac gaggacggcg gcactctcac tgctactcag gacacctccc tccaggacgg
     2581 ctgcctcatc tacaacgtcc aggtcagggg cgagaacttc ccttccaacg gccctgtcat
     2641 gcagaagaag actctcggct gggaggcctc caccgagacc ctctaccccg ctgacggctc
     2701 cctcgagggc aggctcgact gggccctcaa gctcgtcggc ggcggccacc tccactgcag
     2761 ggcggccgcc attctatgct atacggtaca agattgtggc tactatgcaa gctccatagc
     2821 gaatgatctt acactgacct tgtcgaccgt tctgaatgat taacaatggt cgcaagtgtg
     2881 gttgacccac ctgcgggcgc ttcttaacta tctatcatcg gtatgtcaag tatcacaccc
     2941 tttaaacaaa agaataagag cataatacag cagtctcatc atggcaaccc ttcgcgcaat
     3001 gtacaaaaag cagccagaag cccccacgca caagcaataa tgtcgtgttc taccctgcca
     3061 tgaacaagcg tcatggccat tagcgaacgc cccaatattc agccccattc gccaagcaag
     3121 ttgcctcgcc cccttcccag aagatcatgg gcatctcgtc ttcttctaat ttcgcatttt
     3181 gtgaagctcc attcatgcac gtcggcttct ggacgactct tatcatatgc attggcctgc
     3241 tcggtgtgag gatgagccac tcccacatgt ttagacggca ggggcctctt gccacccgtt
     3301 gaatttttgg tgcgaagacg tgcctgggaa cgacctccac tttaaatttc tccgttgttg
     3361 cggtcgctcc atcctcctca catttcttgc ctcctaagtc ccatgtgcca cactcggtga
     3421 aaaacggagt ttgatacgga tgaaatttcc agtgagcttt tcacattctg gaggtttcga
     3481 aaaaaattgt gggattttct gaattaatcc gagaatccgc cctgcccatc aaccggaaag
     3541 aattaaatcc ctcgcacatt cctttctccc accaccttct ttatctctac attctttaac
     3601 caaaacacct aggtgagtac ccaaagtctt ttgacacctt tgaaaatccg cgccagtcgt
     3661 agggcacgta aaacaattct cgctggtggc ttctgcaggt gaagtcgggg cagagtggag
     3721 ctgagcatgg tcgaccaccc acatcattct gtcttgtttt tggcgatgtc ttccatgctt
     3781 ttgccctcat ctgttgcgtc gactgcagag tactgttttg ccactactga gctcaacatg
     3841 ggcttgtgtg tggtgaagaa aaaaatttgg tggctcttga aaattttcaa cccatcggat
     3901 acatgccctt ccaactcaca acgccctaac actttattgt cctatcattg cctcttcgca
     3961 catgctaacg atctccacag aaaacttcaa aatgcctcaa tcctgggaag aactggccgc
     4021 tgataagcgc gcccgcctcg caaaaaccat ccctgatgaa tggaaagtcc agacgctgcc
     4081 tgcggaagac agcgttattg atttcccaaa gaaatcgggg atcctttcag aggccgaact
     4141 gaagatcaca gaggcctccg ctgcagatct tgtgtccaag ctggcggccg gagagttgac
     4201 ctcggtggaa gttacgctag cattctgtaa acgggcagca atcgcccagc agttaacaaa
     4261 ctgcgcccac gagttcttcc ctgacgccgc tctcgcgcag gcaagggaac tcgatgaata
     4321 ctacgcaaag cacaagagac ccgttggtcc actccatggc ctccccatct ctctcaaaga
     4381 ccagcttcga gtcaagggct acgaaacatc aatgggctac atctcatggc taaacaagta
     4441 cgacgaaggg gactcggttc tgacaaccat gctccgcaaa gccggtgccg tcttctacgt
     4501 caagacctct gtcccgcaga ccctgatggt ctgcgagaca gtcaacaaca tcatcgggcg
     4561 caccgtcaac ccacgcaaca agaactggtc gtgcggcggc agttccggtg gtgagggtgc
     4621 gatcgttggg attcgtggtg gcgtcatcgg tgtaggaacg gatatcggtg gctcgattcg
     4681 agtgccggcc gcgttcaact tcctgtacgg tctaaggccg agtcatgggc ggctgccgta
     4741 tgcaaagatg gcgaacagca tggagggtca ggagacggtg cacagcgttg tcgggccgat
     4801 tacgcactct gttgaggacc tccgcctctt caccaaatcc gtcctcggtc aggagccatg
     4861 gaaatacgac tccaaggtca tccccatgcc ctggcgccag tccgagtcgg acattattgc
     4921 ctccaagatc aagaacggcg ggctcaatat cggctactac aacttcgacg gcaatgtcct
     4981 tccacaccct cctatcctgc gcggcgtgga aaccaccgtc gccgcactcg ccaaagccgg
     5041 tcacaccgtg accccgtgga cgccatacaa gcacgatttc ggccacgatc tcatctccca
     5101 tatctacgcg gctgacggca gcgccgacgt aatgcgcgat atcagtgcat ccggcgagcc
     5161 ggcgattcca aatatcaaag acctactgaa cccgaacatc aaagctgtta acatgaacga
     5221 gctctgggac acgcatctcc agaagtggaa ttaccagatg gagtaccttg agaaatggcg
     5281 ggaggctgaa gaaaaggccg ggaaggaact ggacgccatc atcgcgccga ttacgcctac
     5341 cgctgcggta cggcatgacc agttccggta ctatgggtat gcctctgtga tcaacctgct
     5401 ggatttcacg agcgtggttg ttccggttac ctttgcggat aagaacatcg ataagaagaa
     5461 tgagagtttc aaggcggtta gtgagcttga tgccctcgtg caggaagagt atgatccgga
     5521 ggcgtaccat ggggcaccgg ttgcagtgca ggttatcgga cggagactca gtgaagagag
     5581 gacgttggcg attgcagagg aagtggggaa gttgctggga aatgtggtga ctccatagaa
     5641 ggttgagcgc gtaatagtgg gttggtcttg tctctatcca ttcatctgtg agacatactg
     5701 atttcaactt tttcatcaga taataaggat ggtgtgagat aagcaaggct gggttatgag
     5761 gtttgggact agatggtgtt tcgcgtctaa aggtgagtct tcgttggttc aatcacattg
     5821 atctgatcgc tgatcaattg tcagcctctg aatgtgctct tgaagaaaat ggttgtctgt
     5881 aagaatccag aaccaaaaac ggtcatttca agtatgtatc ctccgtgaag ttcctttagc
     5941 tcttgctaat attggacttt agcgtgcttt agccaaagac agggtcgcga ggttaagagg
     6001 atgggccagg aaaaatctaa gttagatttg tcacacaaag aaaaagattg tctcagtttt
     6061 ggttttgttt ggtttgattt atgcatagga aatatttatg atcgaaatca ctcatttcct
     6121 gcttcgtaaa tatcatctac tatatgcttc gtaaatgtcc atctcctaca tattgtggat
     6181 atgataaatg caatcctctc gtctacactc tcggcttatg cctttgtctt ctttgcgctc
     6241 cttttagcct tctttttatt cctagtcttc aagcccatct acggctccaa gaccttcatt
     6301 aaccaccctc ctgacattcc cgactacttc aagcagtcct tccctgaggg cttcacttgg
     6361 gagaggacca ccgtttacga ggacggcggc actctcactg ctactcagga cacctccctc
     6421 caggacggct gcctcatcta caacgtccag gtcaggggcg agaacttccc ttccaacggc
     6481 cctgtcatgc agaagaagac tctcggctgg gaggcctcca ccgagaccct ctaccccgct
     6541 gacggctccc tcgagggcag gctcgactgg gccctcaagc tcgtcggcgg cggccacctc
     6601 cactgcaggc tcgagaccac ttacaggtcc aagaagcccg ctaagaacct caagatgccc
     6661 ggcgtctact tcattgacag gaggctcgag aggatcaagg aggccgacaa cgagacctac
     6721 gtcgagcagc acgaggtcgc tgtcgccagg tactgcgacc tcccttccaa gctcggccac
     6781 aagcttaacg gcatggacga gctctacaag aattcgatat caagcttatc gataccgtcg
     6841 acctcgaggg ggggcccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     6901 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     6961 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     7021 ccttcccaac agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt
     7081 taaaattcgc gttaaatttt tgttaaatca gctcattttt ttaaccaata ggccgaaatc
     7141 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     7201 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     7261 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     7321 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     7381 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     7441 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     7501 ctacagggcg cgtcag
//
