LOCUS       sequence-313811-        7491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recyclable marker for use in Cryptococcus neoformans. Contains the 
            amdS2 counterselectable marker flanked by the mTurquoise2 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 7491)
  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 7491)
  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: pPEE45
FEATURES             Location/Qualifiers
     source          1..7491
                     /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             3981..5627
                     /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(6805..6821)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6806..6822)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(6846..6865)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6847..6865)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6875..6892)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6875..6891)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6884..6906)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(7032..7488)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7170..7191)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7382..7401
                     /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 gctcttcacc ggcgtcgtcc ccatcctcgt
     2281 cgagctcgac ggcgacgtta acggccacaa gttctccgtc tccggcgagg gcgagggcga
     2341 cgccacctac ggcaagctca ccctcaagtt catctgcacc accggcaagc tccccgtccc
     2401 ctggcccacc ctcgtcacca ccctctcctg gggcgtccag tgcttcgccc gttaccccga
     2461 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gagggctacg tccaggagcg
     2521 taccatcttc ttcaaggacg acggcaacta caagacccgt gccgaggtca agttcgaggg
     2581 cgacaccctc gtcaaccgta tcgagctcaa gggcatcgac ttcaaggagg acggcaacat
     2641 cctcggccac aagctcgagt acaactactt ctccgacaac gtctacatca ccgccgacaa
     2701 gcagaagaac ggcatcaagg ccaacttcaa gatccgtcac aacatcgagg gcggccgcca
     2761 ttctatgcta tacggtacaa gattgtggct actatgcaag ctccatagcg aatgatctta
     2821 cactgacctt gtcgaccgtt ctgaatgatt aacaatggtc gcaagtgtgg ttgacccacc
     2881 tgcgggcgct tcttaactat ctatcatcgg tatgtcaagt atcacaccct ttaaacaaaa
     2941 gaataagagc ataatacagc agtctcatca tggcaaccct tcgcgcaatg tacaaaaagc
     3001 agccagaagc ccccacgcac aagcaataat gtcgtgttct accctgccat gaacaagcgt
     3061 catggccatt agcgaacgcc ccaatattca gccccattcg ccaagcaagt tgcctcgccc
     3121 ccttcccaga agatcatggg catctcgtct tcttctaatt tcgcattttg tgaagctcca
     3181 ttcatgcacg tcggcttctg gacgactctt atcatatgca ttggcctgct cggtgtgagg
     3241 atgagccact cccacatgtt tagacggcag gggcctcttg ccacccgttg aatttttggt
     3301 gcgaagacgt gcctgggaac gacctccact ttaaatttct ccgttgttgc ggtcgctcca
     3361 tcctcctcac atttcttgcc tcctaagtcc catgtgccac actcggtgaa aaacggagtt
     3421 tgatacggat gaaatttcca gtgagctttt cacattctgg aggtttcgaa aaaaattgtg
     3481 ggattttctg aattaatccg agaatccgcc ctgcccatca accggaaaga attaaatccc
     3541 tcgcacattc ctttctccca ccaccttctt tatctctaca ttctttaacc aaaacaccta
     3601 ggtgagtacc caaagtcttt tgacaccttt gaaaatccgc gccagtcgta gggcacgtaa
     3661 aacaattctc gctggtggct tctgcaggtg aagtcggggc agagtggagc tgagcatggt
     3721 cgaccaccca catcattctg tcttgttttt ggcgatgtct tccatgcttt tgccctcatc
     3781 tgttgcgtcg actgcagagt actgttttgc cactactgag ctcaacatgg gcttgtgtgt
     3841 ggtgaagaaa aaaatttggt ggctcttgaa aattttcaac ccatcggata catgcccttc
     3901 caactcacaa cgccctaaca ctttattgtc ctatcattgc ctcttcgcac atgctaacga
     3961 tctccacaga aaacttcaaa atgcctcaat cctgggaaga actggccgct gataagcgcg
     4021 cccgcctcgc aaaaaccatc cctgatgaat ggaaagtcca gacgctgcct gcggaagaca
     4081 gcgttattga tttcccaaag aaatcgggga tcctttcaga ggccgaactg aagatcacag
     4141 aggcctccgc tgcagatctt gtgtccaagc tggcggccgg agagttgacc tcggtggaag
     4201 ttacgctagc attctgtaaa cgggcagcaa tcgcccagca gttaacaaac tgcgcccacg
     4261 agttcttccc tgacgccgct ctcgcgcagg caagggaact cgatgaatac tacgcaaagc
     4321 acaagagacc cgttggtcca ctccatggcc tccccatctc tctcaaagac cagcttcgag
     4381 tcaagggcta cgaaacatca atgggctaca tctcatggct aaacaagtac gacgaagggg
     4441 actcggttct gacaaccatg ctccgcaaag ccggtgccgt cttctacgtc aagacctctg
     4501 tcccgcagac cctgatggtc tgcgagacag tcaacaacat catcgggcgc accgtcaacc
     4561 cacgcaacaa gaactggtcg tgcggcggca gttccggtgg tgagggtgcg atcgttggga
     4621 ttcgtggtgg cgtcatcggt gtaggaacgg atatcggtgg ctcgattcga gtgccggccg
     4681 cgttcaactt cctgtacggt ctaaggccga gtcatgggcg gctgccgtat gcaaagatgg
     4741 cgaacagcat ggagggtcag gagacggtgc acagcgttgt cgggccgatt acgcactctg
     4801 ttgaggacct ccgcctcttc accaaatccg tcctcggtca ggagccatgg aaatacgact
     4861 ccaaggtcat ccccatgccc tggcgccagt ccgagtcgga cattattgcc tccaagatca
     4921 agaacggcgg gctcaatatc ggctactaca acttcgacgg caatgtcctt ccacaccctc
     4981 ctatcctgcg cggcgtggaa accaccgtcg ccgcactcgc caaagccggt cacaccgtga
     5041 ccccgtggac gccatacaag cacgatttcg gccacgatct catctcccat atctacgcgg
     5101 ctgacggcag cgccgacgta atgcgcgata tcagtgcatc cggcgagccg gcgattccaa
     5161 atatcaaaga cctactgaac ccgaacatca aagctgttaa catgaacgag ctctgggaca
     5221 cgcatctcca gaagtggaat taccagatgg agtaccttga gaaatggcgg gaggctgaag
     5281 aaaaggccgg gaaggaactg gacgccatca tcgcgccgat tacgcctacc gctgcggtac
     5341 ggcatgacca gttccggtac tatgggtatg cctctgtgat caacctgctg gatttcacga
     5401 gcgtggttgt tccggttacc tttgcggata agaacatcga taagaagaat gagagtttca
     5461 aggcggttag tgagcttgat gccctcgtgc aggaagagta tgatccggag gcgtaccatg
     5521 gggcaccggt tgcagtgcag gttatcggac ggagactcag tgaagagagg acgttggcga
     5581 ttgcagagga agtggggaag ttgctgggaa atgtggtgac tccatagaag gttgagcgcg
     5641 taatagtggg ttggtcttgt ctctatccat tcatctgtga gacatactga tttcaacttt
     5701 ttcatcagat aataaggatg gtgtgagata agcaaggctg ggttatgagg tttgggacta
     5761 gatggtgttt cgcgtctaaa ggtgagtctt cgttggttca atcacattga tctgatcgct
     5821 gatcaattgt cagcctctga atgtgctctt gaagaaaatg gttgtctgta agaatccaga
     5881 accaaaaacg gtcatttcaa gtatgtatcc tccgtgaagt tcctttagct cttgctaata
     5941 ttggacttta gcgtgcttta gccaaagaca gggtcgcgag gttaagagga tgggccagga
     6001 aaaatctaag ttagatttgt cacacaaaga aaaagattgt ctcagttttg gttttgtttg
     6061 gtttgattta tgcataggaa atatttatga tcgaaatcac tcatttcctg cttcgtaaat
     6121 atcatctact atatgcttcg taaatgtcca tctcctacat attgtggata tgataaatgc
     6181 aatcctctcg tctacactct cggcttatgc ctttgtcttc tttgcgctcc ttttagcctt
     6241 ctttttattc ctagtcttca agcccatcgg gcgtccagtg cttcgcccgt taccccgacc
     6301 acatgaagca gcacgacttc ttcaagtccg ccatgcccga gggctacgtc caggagcgta
     6361 ccatcttctt caaggacgac ggcaactaca agacccgtgc cgaggtcaag ttcgagggcg
     6421 acaccctcgt caaccgtatc gagctcaagg gcatcgactt caaggaggac ggcaacatcc
     6481 tcggccacaa gctcgagtac aactacttct ccgacaacgt ctacatcacc gccgacaagc
     6541 agaagaacgg catcaaggcc aacttcaaga tccgtcacaa catcgaggac ggcggcgtcc
     6601 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtc ctcctccccg
     6661 acaaccacta cctctccacc cagtccaagc tctccaagga ccccaacgag aagcgtgacc
     6721 acatggtcct cctcgagttc gtcaccgccg ccggcatcac tctcggcatg gacgagctct
     6781 acaagaattc gatatcaagc ttatcgatac cgtcgacctc gagggggggc ccggtaccca
     6841 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     6901 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     6961 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     7021 atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     7081 aatcagctca tttttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     7141 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     7201 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     7261 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     7321 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     7381 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     7441 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca g
//
