LOCUS       sequence-324126-        7486 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recyclable marker for use in Cryptococcus neoformans. Contains the 
            amdS2 counterselectable marker flanked by the Citrine 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 7486)
  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 7486)
  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: pPEE42
FEATURES             Location/Qualifiers
     source          1..7486
                     /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             3977..5623
                     /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(6800..6816)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6801..6817)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(6841..6860)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6842..6860)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6870..6887)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6870..6886)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6879..6901)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(7027..7483)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7165..7186)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7377..7396
                     /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 ccttcggcta cggcctcatg 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 acaactacaa ctcccacaac gtctacatca tggccgacaa
     2701 gcagaagaac ggcatcaagg tcaacttcaa gatccgtcac aacatcgcgg ccgccattct
     2761 atgctatacg gtacaagatt gtggctacta tgcaagctcc atagcgaatg atcttacact
     2821 gaccttgtcg accgttctga atgattaaca atggtcgcaa gtgtggttga cccacctgcg
     2881 ggcgcttctt aactatctat catcggtatg tcaagtatca caccctttaa acaaaagaat
     2941 aagagcataa tacagcagtc tcatcatggc aacccttcgc gcaatgtaca aaaagcagcc
     3001 agaagccccc acgcacaagc aataatgtcg tgttctaccc tgccatgaac aagcgtcatg
     3061 gccattagcg aacgccccaa tattcagccc cattcgccaa gcaagttgcc tcgccccctt
     3121 cccagaagat catgggcatc tcgtcttctt ctaatttcgc attttgtgaa gctccattca
     3181 tgcacgtcgg cttctggacg actcttatca tatgcattgg cctgctcggt gtgaggatga
     3241 gccactccca catgtttaga cggcaggggc ctcttgccac ccgttgaatt tttggtgcga
     3301 agacgtgcct gggaacgacc tccactttaa atttctccgt tgttgcggtc gctccatcct
     3361 cctcacattt cttgcctcct aagtcccatg tgccacactc ggtgaaaaac ggagtttgat
     3421 acggatgaaa tttccagtga gcttttcaca ttctggaggt ttcgaaaaaa attgtgggat
     3481 tttctgaatt aatccgagaa tccgccctgc ccatcaaccg gaaagaatta aatccctcgc
     3541 acattccttt ctcccaccac cttctttatc tctacattct ttaaccaaaa cacctaggtg
     3601 agtacccaaa gtcttttgac acctttgaaa atccgcgcca gtcgtagggc acgtaaaaca
     3661 attctcgctg gtggcttctg caggtgaagt cggggcagag tggagctgag catggtcgac
     3721 cacccacatc attctgtctt gtttttggcg atgtcttcca tgcttttgcc ctcatctgtt
     3781 gcgtcgactg cagagtactg ttttgccact actgagctca acatgggctt gtgtgtggtg
     3841 aagaaaaaaa tttggtggct cttgaaaatt ttcaacccat cggatacatg cccttccaac
     3901 tcacaacgcc ctaacacttt attgtcctat cattgcctct tcgcacatgc taacgatctc
     3961 cacagaaaac ttcaaaatgc ctcaatcctg ggaagaactg gccgctgata agcgcgcccg
     4021 cctcgcaaaa accatccctg atgaatggaa agtccagacg ctgcctgcgg aagacagcgt
     4081 tattgatttc ccaaagaaat cggggatcct ttcagaggcc gaactgaaga tcacagaggc
     4141 ctccgctgca gatcttgtgt ccaagctggc ggccggagag ttgacctcgg tggaagttac
     4201 gctagcattc tgtaaacggg cagcaatcgc ccagcagtta acaaactgcg cccacgagtt
     4261 cttccctgac gccgctctcg cgcaggcaag ggaactcgat gaatactacg caaagcacaa
     4321 gagacccgtt ggtccactcc atggcctccc catctctctc aaagaccagc ttcgagtcaa
     4381 gggctacgaa acatcaatgg gctacatctc atggctaaac aagtacgacg aaggggactc
     4441 ggttctgaca accatgctcc gcaaagccgg tgccgtcttc tacgtcaaga cctctgtccc
     4501 gcagaccctg atggtctgcg agacagtcaa caacatcatc gggcgcaccg tcaacccacg
     4561 caacaagaac tggtcgtgcg gcggcagttc cggtggtgag ggtgcgatcg ttgggattcg
     4621 tggtggcgtc atcggtgtag gaacggatat cggtggctcg attcgagtgc cggccgcgtt
     4681 caacttcctg tacggtctaa ggccgagtca tgggcggctg ccgtatgcaa agatggcgaa
     4741 cagcatggag ggtcaggaga cggtgcacag cgttgtcggg ccgattacgc actctgttga
     4801 ggacctccgc ctcttcacca aatccgtcct cggtcaggag ccatggaaat acgactccaa
     4861 ggtcatcccc atgccctggc gccagtccga gtcggacatt attgcctcca agatcaagaa
     4921 cggcgggctc aatatcggct actacaactt cgacggcaat gtccttccac accctcctat
     4981 cctgcgcggc gtggaaacca ccgtcgccgc actcgccaaa gccggtcaca ccgtgacccc
     5041 gtggacgcca tacaagcacg atttcggcca cgatctcatc tcccatatct acgcggctga
     5101 cggcagcgcc gacgtaatgc gcgatatcag tgcatccggc gagccggcga ttccaaatat
     5161 caaagaccta ctgaacccga acatcaaagc tgttaacatg aacgagctct gggacacgca
     5221 tctccagaag tggaattacc agatggagta ccttgagaaa tggcgggagg ctgaagaaaa
     5281 ggccgggaag gaactggacg ccatcatcgc gccgattacg cctaccgctg cggtacggca
     5341 tgaccagttc cggtactatg ggtatgcctc tgtgatcaac ctgctggatt tcacgagcgt
     5401 ggttgttccg gttacctttg cggataagaa catcgataag aagaatgaga gtttcaaggc
     5461 ggttagtgag cttgatgccc tcgtgcagga agagtatgat ccggaggcgt accatggggc
     5521 accggttgca gtgcaggtta tcggacggag actcagtgaa gagaggacgt tggcgattgc
     5581 agaggaagtg gggaagttgc tgggaaatgt ggtgactcca tagaaggttg agcgcgtaat
     5641 agtgggttgg tcttgtctct atccattcat ctgtgagaca tactgatttc aactttttca
     5701 tcagataata aggatggtgt gagataagca aggctgggtt atgaggtttg ggactagatg
     5761 gtgtttcgcg tctaaaggtg agtcttcgtt ggttcaatca cattgatctg atcgctgatc
     5821 aattgtcagc ctctgaatgt gctcttgaag aaaatggttg tctgtaagaa tccagaacca
     5881 aaaacggtca tttcaagtat gtatcctccg tgaagttcct ttagctcttg ctaatattgg
     5941 actttagcgt gctttagcca aagacagggt cgcgaggtta agaggatggg ccaggaaaaa
     6001 tctaagttag atttgtcaca caaagaaaaa gattgtctca gttttggttt tgtttggttt
     6061 gatttatgca taggaaatat ttatgatcga aatcactcat ttcctgcttc gtaaatatca
     6121 tctactatat gcttcgtaaa tgtccatctc ctacatattg tggatatgat aaatgcaatc
     6181 ctctcgtcta cactctcggc ttatgccttt gtcttctttg cgctcctttt agccttcttt
     6241 ttattcctag tcttcaagcc catcggcctc atgtgcttcg cccgttaccc cgaccacatg
     6301 aagcagcacg acttcttcaa gtccgccatg cccgagggct acgtccagga gcgtaccatc
     6361 ttcttcaagg acgacggcaa ctacaagacc cgtgccgagg tcaagttcga gggcgacacc
     6421 ctcgtcaacc gtatcgagct caagggcatc gacttcaagg aggacggcaa catcctcggc
     6481 cacaagctcg agtacaacta caactcccac aacgtctaca tcatggccga caagcagaag
     6541 aacggcatca aggtcaactt caagatccgt cacaacatcg aggacggctc cgtccagctc
     6601 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtcctcct ccccgacaac
     6661 cactacctct cctaccagtc cgccctctcc aaggacccca acgagaagcg tgaccacatg
     6721 gtcctcctcg agttcgtcac cgccgccggc atcactctcg gcatggacga gctttacaag
     6781 aattcgatat caagcttatc gataccgtcg acctcgaggg ggggcccggt acccaattcg
     6841 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     6901 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     6961 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     7021 gaatggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     7081 gctcattttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     7141 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     7201 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     7261 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     7321 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     7381 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     7441 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcag
//
