LOCUS       sequence-313810-        7468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recyclable marker for use in Cryptococcus neoformans. Contains the 
            amdS2 counterselectable marker flanked by the mTFP1 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 7468)
  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 7468)
  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: pPEE44
FEATURES             Location/Qualifiers
     source          1..7468
                     /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             3968..5614
                     /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(6782..6798)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6783..6799)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(6823..6842)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6824..6842)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6852..6869)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6852..6868)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6861..6883)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(7009..7465)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7147..7168)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7359..7378
                     /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 gaccactatg ggcgttatca agcccgacat
     2281 gaagatcaag ctcaagatgg agggcaacgt caacggccac gccttcgtca tcgagggcga
     2341 gggcgagggc aagccctacg acggcaccaa caccatcaac ctcgaggtca aggagggtgc
     2401 ccccctcccc ttctcctacg acattctcac caccgccttc gcctacggca acagggcctt
     2461 caccaagtac cccgacgaca tccccaacta cttcaagcag tccttccccg agggctactc
     2521 ttgggagcgt accatgacct tcgaggacaa gggcatcgtc aaggtcaagt ccgacatctc
     2581 catggaggag gactccttca tctacgagat ccacctcaag ggcgagaact tcccccccaa
     2641 cggccccgtc atgcagaaga agaccaccgg ctgggacgcc tccaccgaga ggatgtacgt
     2701 ccgtgacggc gtcctcaagg gcgacgtcaa gcacaaggcg gccgccattc tatgctatac
     2761 ggtacaagat tgtggctact atgcaagctc catagcgaat gatcttacac tgaccttgtc
     2821 gaccgttctg aatgattaac aatggtcgca agtgtggttg acccacctgc gggcgcttct
     2881 taactatcta tcatcggtat gtcaagtatc acacccttta aacaaaagaa taagagcata
     2941 atacagcagt ctcatcatgg caacccttcg cgcaatgtac aaaaagcagc cagaagcccc
     3001 cacgcacaag caataatgtc gtgttctacc ctgccatgaa caagcgtcat ggccattagc
     3061 gaacgcccca atattcagcc ccattcgcca agcaagttgc ctcgccccct tcccagaaga
     3121 tcatgggcat ctcgtcttct tctaatttcg cattttgtga agctccattc atgcacgtcg
     3181 gcttctggac gactcttatc atatgcattg gcctgctcgg tgtgaggatg agccactccc
     3241 acatgtttag acggcagggg cctcttgcca cccgttgaat ttttggtgcg aagacgtgcc
     3301 tgggaacgac ctccacttta aatttctccg ttgttgcggt cgctccatcc tcctcacatt
     3361 tcttgcctcc taagtcccat gtgccacact cggtgaaaaa cggagtttga tacggatgaa
     3421 atttccagtg agcttttcac attctggagg tttcgaaaaa aattgtggga ttttctgaat
     3481 taatccgaga atccgccctg cccatcaacc ggaaagaatt aaatccctcg cacattcctt
     3541 tctcccacca ccttctttat ctctacattc tttaaccaaa acacctaggt gagtacccaa
     3601 agtcttttga cacctttgaa aatccgcgcc agtcgtaggg cacgtaaaac aattctcgct
     3661 ggtggcttct gcaggtgaag tcggggcaga gtggagctga gcatggtcga ccacccacat
     3721 cattctgtct tgtttttggc gatgtcttcc atgcttttgc cctcatctgt tgcgtcgact
     3781 gcagagtact gttttgccac tactgagctc aacatgggct tgtgtgtggt gaagaaaaaa
     3841 atttggtggc tcttgaaaat tttcaaccca tcggatacat gcccttccaa ctcacaacgc
     3901 cctaacactt tattgtccta tcattgcctc ttcgcacatg ctaacgatct ccacagaaaa
     3961 cttcaaaatg cctcaatcct gggaagaact ggccgctgat aagcgcgccc gcctcgcaaa
     4021 aaccatccct gatgaatgga aagtccagac gctgcctgcg gaagacagcg ttattgattt
     4081 cccaaagaaa tcggggatcc tttcagaggc cgaactgaag atcacagagg cctccgctgc
     4141 agatcttgtg tccaagctgg cggccggaga gttgacctcg gtggaagtta cgctagcatt
     4201 ctgtaaacgg gcagcaatcg cccagcagtt aacaaactgc gcccacgagt tcttccctga
     4261 cgccgctctc gcgcaggcaa gggaactcga tgaatactac gcaaagcaca agagacccgt
     4321 tggtccactc catggcctcc ccatctctct caaagaccag cttcgagtca agggctacga
     4381 aacatcaatg ggctacatct catggctaaa caagtacgac gaaggggact cggttctgac
     4441 aaccatgctc cgcaaagccg gtgccgtctt ctacgtcaag acctctgtcc cgcagaccct
     4501 gatggtctgc gagacagtca acaacatcat cgggcgcacc gtcaacccac gcaacaagaa
     4561 ctggtcgtgc ggcggcagtt ccggtggtga gggtgcgatc gttgggattc gtggtggcgt
     4621 catcggtgta ggaacggata tcggtggctc gattcgagtg ccggccgcgt tcaacttcct
     4681 gtacggtcta aggccgagtc atgggcggct gccgtatgca aagatggcga acagcatgga
     4741 gggtcaggag acggtgcaca gcgttgtcgg gccgattacg cactctgttg aggacctccg
     4801 cctcttcacc aaatccgtcc tcggtcagga gccatggaaa tacgactcca aggtcatccc
     4861 catgccctgg cgccagtccg agtcggacat tattgcctcc aagatcaaga acggcgggct
     4921 caatatcggc tactacaact tcgacggcaa tgtccttcca caccctccta tcctgcgcgg
     4981 cgtggaaacc accgtcgccg cactcgccaa agccggtcac accgtgaccc cgtggacgcc
     5041 atacaagcac gatttcggcc acgatctcat ctcccatatc tacgcggctg acggcagcgc
     5101 cgacgtaatg cgcgatatca gtgcatccgg cgagccggcg attccaaata tcaaagacct
     5161 actgaacccg aacatcaaag ctgttaacat gaacgagctc tgggacacgc atctccagaa
     5221 gtggaattac cagatggagt accttgagaa atggcgggag gctgaagaaa aggccgggaa
     5281 ggaactggac gccatcatcg cgccgattac gcctaccgct gcggtacggc atgaccagtt
     5341 ccggtactat gggtatgcct ctgtgatcaa cctgctggat ttcacgagcg tggttgttcc
     5401 ggttaccttt gcggataaga acatcgataa gaagaatgag agtttcaagg cggttagtga
     5461 gcttgatgcc ctcgtgcagg aagagtatga tccggaggcg taccatgggg caccggttgc
     5521 agtgcaggtt atcggacgga gactcagtga agagaggacg ttggcgattg cagaggaagt
     5581 ggggaagttg ctgggaaatg tggtgactcc atagaaggtt gagcgcgtaa tagtgggttg
     5641 gtcttgtctc tatccattca tctgtgagac atactgattt caactttttc atcagataat
     5701 aaggatggtg tgagataagc aaggctgggt tatgaggttt gggactagat ggtgtttcgc
     5761 gtctaaaggt gagtcttcgt tggttcaatc acattgatct gatcgctgat caattgtcag
     5821 cctctgaatg tgctcttgaa gaaaatggtt gtctgtaaga atccagaacc aaaaacggtc
     5881 atttcaagta tgtatcctcc gtgaagttcc tttagctctt gctaatattg gactttagcg
     5941 tgctttagcc aaagacaggg tcgcgaggtt aagaggatgg gccaggaaaa atctaagtta
     6001 gatttgtcac acaaagaaaa agattgtctc agttttggtt ttgtttggtt tgatttatgc
     6061 ataggaaata tttatgatcg aaatcactca tttcctgctt cgtaaatatc atctactata
     6121 tgcttcgtaa atgtccatct cctacatatt gtggatatga taaatgcaat cctctcgtct
     6181 acactctcgg cttatgcctt tgtcttcttt gcgctccttt tagccttctt tttattccta
     6241 gtcttcaagc ccatcaccgc cttcgcctac ggcaacaggg ccttcaccaa gtaccccgac
     6301 gacatcccca actacttcaa gcagtccttc cccgagggct actcttggga gcgtaccatg
     6361 accttcgagg acaagggcat cgtcaaggtc aagtccgaca tctccatgga ggaggactcc
     6421 ttcatctacg agatccacct caagggcgag aacttccccc ccaacggccc cgtcatgcag
     6481 aagaagacca ccggctggga cgcctccacc gagaggatgt acgtccgtga cggcgtcctc
     6541 aagggcgacg tcaagcacaa gctcctcctc gagggcggcg gccaccaccg tgttgacttc
     6601 aagaccatct acagggccaa gaaggccgtc aagctccccg actaccactt cgtcgaccac
     6661 cgtatcgaga tcctcaacca cgacaaggac tacaacaagg tcaccgttta cgagtccgcc
     6721 gtcgcccgta actccaccga cggcatggac gagctctaca agaattcgat atcaagctta
     6781 tcgataccgt cgacctcgag ggggggcccg gtacccaatt cgccctatag tgagtcgtat
     6841 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     6901 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     6961 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggaa attgtaagcg
     7021 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt ttttaaccaa
     7081 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     7141 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     7201 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     7261 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     7321 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     7381 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     7441 cttaatgcgc cgctacaggg cgcgtcag
//
