LOCUS       sequence-297721-        7477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recyclable marker for use in Cryptococcus neoformans. Contains the 
            amdS2 counterselectable marker flanked by the mNeonGreen 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 7477)
  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 7477)
  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: pPEE47
FEATURES             Location/Qualifiers
     source          1..7477
                     /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"
     regulatory      2486..2495
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3972..5618
                     /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"
     regulatory      6315..6324
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6791..6807)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6792..6808)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(6832..6851)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6833..6851)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6861..6878)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6861..6877)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6870..6892)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(7018..7474)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7156..7177)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7368..7387
                     /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 ggacaacatg gcctctctcc ctgccactca
     2281 cgagttgcac atcttcggct ccatcaacgg tgtcgacttc gacatggtcg gtcagggcac
     2341 cggcaaccct aacgacggtt acgaggagtt gaaccttaag tccaccaagg gtgacctcca
     2401 gttctccccc tggattcttg tccctcacat cggctacggc ttccaccagt accttcccta
     2461 ccctgacggc atgtcccctt tccaggccgc catggttgac ggctccggtt accaggtcca
     2521 ccgtactatg cagttcgagg acggtgcctc ccttactgtt aactaccgtt acacctacga
     2581 gggttcccac atcaagggtg aggcccaggt caagggcact ggtttccctg ctgacggtcc
     2641 tgtcatgacc aactccctta ccgctgccga ctggtgcagg tccaagaaga cttaccccaa
     2701 cgacaagacc atcatctcca ccttcaagtg gtcctacacc agcggccgcc attctatgct
     2761 atacggtaca agattgtggc tactatgcaa gctccatagc gaatgatctt acactgacct
     2821 tgtcgaccgt tctgaatgat taacaatggt cgcaagtgtg gttgacccac ctgcgggcgc
     2881 ttcttaacta tctatcatcg gtatgtcaag tatcacaccc tttaaacaaa agaataagag
     2941 cataatacag cagtctcatc atggcaaccc ttcgcgcaat gtacaaaaag cagccagaag
     3001 cccccacgca caagcaataa tgtcgtgttc taccctgcca tgaacaagcg tcatggccat
     3061 tagcgaacgc cccaatattc agccccattc gccaagcaag ttgcctcgcc cccttcccag
     3121 aagatcatgg gcatctcgtc ttcttctaat ttcgcatttt gtgaagctcc attcatgcac
     3181 gtcggcttct ggacgactct tatcatatgc attggcctgc tcggtgtgag gatgagccac
     3241 tcccacatgt ttagacggca ggggcctctt gccacccgtt gaatttttgg tgcgaagacg
     3301 tgcctgggaa cgacctccac tttaaatttc tccgttgttg cggtcgctcc atcctcctca
     3361 catttcttgc ctcctaagtc ccatgtgcca cactcggtga aaaacggagt ttgatacgga
     3421 tgaaatttcc agtgagcttt tcacattctg gaggtttcga aaaaaattgt gggattttct
     3481 gaattaatcc gagaatccgc cctgcccatc aaccggaaag aattaaatcc ctcgcacatt
     3541 cctttctccc accaccttct ttatctctac attctttaac caaaacacct aggtgagtac
     3601 ccaaagtctt ttgacacctt tgaaaatccg cgccagtcgt agggcacgta aaacaattct
     3661 cgctggtggc ttctgcaggt gaagtcgggg cagagtggag ctgagcatgg tcgaccaccc
     3721 acatcattct gtcttgtttt tggcgatgtc ttccatgctt ttgccctcat ctgttgcgtc
     3781 gactgcagag tactgttttg ccactactga gctcaacatg ggcttgtgtg tggtgaagaa
     3841 aaaaatttgg tggctcttga aaattttcaa cccatcggat acatgccctt ccaactcaca
     3901 acgccctaac actttattgt cctatcattg cctcttcgca catgctaacg atctccacag
     3961 aaaacttcaa aatgcctcaa tcctgggaag aactggccgc tgataagcgc gcccgcctcg
     4021 caaaaaccat ccctgatgaa tggaaagtcc agacgctgcc tgcggaagac agcgttattg
     4081 atttcccaaa gaaatcgggg atcctttcag aggccgaact gaagatcaca gaggcctccg
     4141 ctgcagatct tgtgtccaag ctggcggccg gagagttgac ctcggtggaa gttacgctag
     4201 cattctgtaa acgggcagca atcgcccagc agttaacaaa ctgcgcccac gagttcttcc
     4261 ctgacgccgc tctcgcgcag gcaagggaac tcgatgaata ctacgcaaag cacaagagac
     4321 ccgttggtcc actccatggc ctccccatct ctctcaaaga ccagcttcga gtcaagggct
     4381 acgaaacatc aatgggctac atctcatggc taaacaagta cgacgaaggg gactcggttc
     4441 tgacaaccat gctccgcaaa gccggtgccg tcttctacgt caagacctct gtcccgcaga
     4501 ccctgatggt ctgcgagaca gtcaacaaca tcatcgggcg caccgtcaac ccacgcaaca
     4561 agaactggtc gtgcggcggc agttccggtg gtgagggtgc gatcgttggg attcgtggtg
     4621 gcgtcatcgg tgtaggaacg gatatcggtg gctcgattcg agtgccggcc gcgttcaact
     4681 tcctgtacgg tctaaggccg agtcatgggc ggctgccgta tgcaaagatg gcgaacagca
     4741 tggagggtca ggagacggtg cacagcgttg tcgggccgat tacgcactct gttgaggacc
     4801 tccgcctctt caccaaatcc gtcctcggtc aggagccatg gaaatacgac tccaaggtca
     4861 tccccatgcc ctggcgccag tccgagtcgg acattattgc ctccaagatc aagaacggcg
     4921 ggctcaatat cggctactac aacttcgacg gcaatgtcct tccacaccct cctatcctgc
     4981 gcggcgtgga aaccaccgtc gccgcactcg ccaaagccgg tcacaccgtg accccgtgga
     5041 cgccatacaa gcacgatttc ggccacgatc tcatctccca tatctacgcg gctgacggca
     5101 gcgccgacgt aatgcgcgat atcagtgcat ccggcgagcc ggcgattcca aatatcaaag
     5161 acctactgaa cccgaacatc aaagctgtta acatgaacga gctctgggac acgcatctcc
     5221 agaagtggaa ttaccagatg gagtaccttg agaaatggcg ggaggctgaa gaaaaggccg
     5281 ggaaggaact ggacgccatc atcgcgccga ttacgcctac cgctgcggta cggcatgacc
     5341 agttccggta ctatgggtat gcctctgtga tcaacctgct ggatttcacg agcgtggttg
     5401 ttccggttac ctttgcggat aagaacatcg ataagaagaa tgagagtttc aaggcggtta
     5461 gtgagcttga tgccctcgtg caggaagagt atgatccgga ggcgtaccat ggggcaccgg
     5521 ttgcagtgca ggttatcgga cggagactca gtgaagagag gacgttggcg attgcagagg
     5581 aagtggggaa gttgctggga aatgtggtga ctccatagaa ggttgagcgc gtaatagtgg
     5641 gttggtcttg tctctatcca ttcatctgtg agacatactg atttcaactt tttcatcaga
     5701 taataaggat ggtgtgagat aagcaaggct gggttatgag gtttgggact agatggtgtt
     5761 tcgcgtctaa aggtgagtct tcgttggttc aatcacattg atctgatcgc tgatcaattg
     5821 tcagcctctg aatgtgctct tgaagaaaat ggttgtctgt aagaatccag aaccaaaaac
     5881 ggtcatttca agtatgtatc ctccgtgaag ttcctttagc tcttgctaat attggacttt
     5941 agcgtgcttt agccaaagac agggtcgcga ggttaagagg atgggccagg aaaaatctaa
     6001 gttagatttg tcacacaaag aaaaagattg tctcagtttt ggttttgttt ggtttgattt
     6061 atgcatagga aatatttatg atcgaaatca ctcatttcct gcttcgtaaa tatcatctac
     6121 tatatgcttc gtaaatgtcc atctcctaca tattgtggat atgataaatg caatcctctc
     6181 gtctacactc tcggcttatg cctttgtctt ctttgcgctc cttttagcct tctttttatt
     6241 cctagtcttc aagcccatcc ggctacggct tccaccagta ccttccctac cctgacggca
     6301 tgtccccttt ccaggccgcc atggttgacg gctccggtta ccaggtccac cgtactatgc
     6361 agttcgagga cggtgcctcc cttactgtta actaccgtta cacctacgag ggttcccaca
     6421 tcaagggtga ggcccaggtc aagggcactg gtttccctgc tgacggtcct gtcatgacca
     6481 actcccttac cgctgccgac tggtgcaggt ccaagaagac ttaccccaac gacaagacca
     6541 tcatctccac cttcaagtgg tcctacacca ctggtaacgg caagcgttac cgatccactg
     6601 cccgaaccac ctacaccttc gccaagccta tggccgctaa ctaccttaag aaccagccca
     6661 tgtacgtttt ccgtaagacc gagctcaagc actccaagac cgagctcaac ttcaaggagt
     6721 ggcagaaggc cttcaccgac gtcatgggca tggacgagct ttacaagtaa gaattcgata
     6781 tcaagcttat cgataccgtc gacctcgagg gggggcccgg tacccaattc gccctatagt
     6841 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     6901 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     6961 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa
     7021 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     7081 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     7141 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     7201 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     7261 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     7321 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     7381 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     7441 cccgccgcgc ttaatgcgcc gctacagggc gcgtcag
//
