LOCUS       sequence-292470-        8409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluoroescent marker for use in Cryptococcus neoformans. Contains a 
            codon optimised Citrine marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8409)
  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 8409)
  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: pGWKS5
FEATURES             Location/Qualifiers
     source          1..8409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1025..1588)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="TLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFTLR
                     EVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRRLT
                     VEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTLCG
                     LDTALYDGTASDGEQALYMSMPCP"
     primer_bind     2696..2718
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2710..2727
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2711..2727
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2737..2756
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2737..2755
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4313..4332)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(5416..5436)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5416..5434)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5455..5471)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5455..5471)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5468..5490)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5479..5495
                     /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)."
     promoter        complement(join(5503..5509,5510..5527,5528..5533))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5548..5569
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5686..5703)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5857..6445)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5937..5956)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6616..7407,7408..7476))
                     /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     7239..7258
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7477..7581)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 accaaaccag tctgtgccaa gtggattgct tatgcatcga gtggcgagcc cactgacctc
       61 tgggcccctt ttttgaacta taaaaagcgg ccaagtctga tttcggtctc acaatcatca
      121 acatcgactt ctcttttcgc ctcgatcaac ctaaacaacg gtcactcagc catgaactgg
      181 acaatggaat atttcgcctt gccttggatc tttggcctct agactcaatc aacgtcagga
      241 taaaggtgag cataggttgc tcactcactg tagccttttt ttcccttctt cttggtccag
      301 atggttccca aggctctata aagtgtatct gtcagttcca cttccttact ccgaagcgat
      361 tcagagttgt aaagtgcgtc tgaccccctg attatatctc aaagtccctt cacaaattca
      421 gacatgatgc gataaagtgc cggattgtca ttcgcgtctt gtgactgtat agaaagtgcc
      481 ggcatcctct tttcttttcc tctttggccg agcgactcag agttgtgaag cctgctggta
      541 tctgtctgtt cccttgccct gataaagaat atcgaagagc atttctttga ttcacaatac
      601 gttgacgaat ggtgtgatgg atcctaagca gcgccagccc tttgaacttg tgtcgaaaaa
      661 aaaaaaaaaa cagcatatga tacgtaaagg gaagaaaagg atggagctga ctgttttgac
      721 aggcataatt ttgagcgtgc ttcattggcc ggtttatctg tattaacacg gaagagatgt
      781 agaaactagc ttcctggttt cagagacaag gagccatgaa gatcctgagg attcaacggc
      841 gttgctgagg gtgggaagga tggtattgtt gtctgtagat agattaatgg gcatataatg
      901 tcaaaaaaga tgattgacac gatgtctgtc tcgaattcgc agtgtgctaa aattgtatca
      961 cttctctaat atgtatatat acaccctcta aggaaaatta accccttacc gccttcacga
     1021 attcttaggg gcagggcatg ctcatgtaga gcgcctgctc gccgtccgag gcggtgccgt
     1081 cgtacagggc ggtgtccagg ccgcagaggg tgaaccccat ccgccggtac gcgtggatag
     1141 ccggtgcgtt aacgttggtg acctccagcc agaggtgccc ggcgccccgc tcgcgggcga
     1201 actccgtcgc gagccccatc aacgcgcgcc cgaccccgtg cccccggtgc tccggggcga
     1261 cctcgatgtc ctcgacggtc agccggcggt tccagccgga gtacgagacg accacgaagc
     1321 ccgccaggtc gccgtcgtcc ccgtacgcga cgaacgtccg ggagtccggg tcgccgtcct
     1381 ccccgtcgtc cgattcgtcg tccgattcgt cgtcggggaa caccttggtc aggggcgggt
     1441 ccaccggcac ctcccgcagg gtgaagccgt ccccggtggc ggtgacgcgg aagacggtgt
     1501 cggtggtgaa ggacccatcc agtgcctcga tggcctcggc gtcccccggg acactggtgc
     1561 ggtaccggta agccgtgtcg tcaagagtgg cggccgccat agacatgttg ggcgagttta
     1621 ctaatggaaa aagacaatta gcaaagaggc tgcatattag gagacgcggt gaagattggg
     1681 gaggacaata gggtccgtgc accaaaaagt cctgacggca gctctgttca acgcacatcc
     1741 atctcttcca gctatgcgtg tggcaccagc acggatggat tgttgcacca aaaggacaag
     1801 gcgagatgga tgcaggtcga ggcgatgcgg tgctatcggc cctcgttgtc gaacgccacg
     1861 tcgtcctctc ggcgaccgtc aatcctggaa ggaaatgacc aagcgtgcac ccagcatcca
     1921 gcatccaaca tccagcaccc agcacctagc acccatgatc caatggccgg catgcagatc
     1981 agcgtgggga gagcaccatc tagcagcccc tggcaggccc agcattgtgc tcggatgtgc
     2041 gataggcggt gggtgcgata aggtggatta gtggaggatg gaggactgag tggcgggcaa
     2101 gggaaggggg cgggaagatg actcaccttg ggcagtgggt gggggatgcg gtatagggga
     2161 tgtatgtgga tgctggcgga ggatagaagc tgtaagctga gctggtgcca acggtgacgc
     2221 tgtgagagtg gtctcggcga ccctgacggc gactcggcgt taccccgtgt ggcacaccgg
     2281 acgcgcgtcg agtgacgttg ctgctattgt tgttaccatc atcctctcct ccgacaacca
     2341 tacactcagt gcacaccatg ccactcgaat cctgcatgct tatgtgagtc ctccccttcc
     2401 cgtgcgccgc tctccagctc acatcctcgc agccatggac tcaaccctat ctcggtctat
     2461 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa atgagctgat
     2521 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt ccattcgcca
     2581 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     2641 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     2701 tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa
     2761 ttgggtaccg ggccccccct cgagcattct atgctatacg gtacaagatt gtggctacta
     2821 tgcaagctcc atagcgaatg atcttacact gaccttgtcg accgttctga atgattaaca
     2881 atggtcgcaa gtgtggttga cccacctgcg ggcgcttctt aactatctat catcggtatg
     2941 tcaagtatca ctccctttaa acaaaagaat aagagcataa tacagcagtc tcatcatggc
     3001 aacccttcgc gcaatgtaca aaaagcagcc agaagccccc acgcacaagc aataatgtcg
     3061 tgttctaccc tgccatgaac aagcgtcatg gccattagcg aacgccccaa tattcagccc
     3121 cattcgccaa gcaagttgcc tcgccccctt cccagaagat catgggcatc tcgtcttctt
     3181 ctaatttcgc attttgtgaa gctccattca tgcacgtcgg cttctggacg actcttatca
     3241 tatgcattgg cctgctcggt gtgaggatga gccactccca catgtttaga cggcaggggc
     3301 ctcttgccac ccgttgaatt tttggtgcga agacgtgcct gggaacgacc tccactttaa
     3361 atttctccgt tgttgcggtc gctccatcct cctcacattt cttgcctcct aagtcccatg
     3421 tgccacactc ggtgaaaaac ggagtttgat acggatgaaa tttccagtga gcttttcaca
     3481 ttctggaggt ttcgaaaaaa attgtgggat tttctgaatt aatccgagaa tccgccctgc
     3541 ccatcaaccg gaaagaatta aatccctcgc acattccttt ctcccaccac cttctttatc
     3601 tctacattct ttaaccaaaa cacctaggtg agtacccaaa gtcttttgac acctttgaaa
     3661 atccgcgcca gtcgtagggc acgtaaaaca attctcgctg gtggcttctg caggtgaagt
     3721 cggggcagag tggagctgag catggtcgac cacccacatc attctgtctt gtttttggcg
     3781 atgtcttcca tgcttttgcc ctcatctgtt gcgtcgactg cagagtactg ttttgccact
     3841 actgagctca acatgggctt gtgtgtggtg aagaaaaaaa tttggtggct cttgaaaatt
     3901 ttcaacccat cggatacatg cccttccaac tcacaacgcc ctaacacttt attgtcctat
     3961 cattgcctct tcgcacatgc taacgatctc cacagaaaac ttcaaaatgg tctccaaggg
     4021 cgaggagctc ttcaccggcg tcgtccccat cctcgtcgag ctcgacggcg acgttaacgg
     4081 ccacaagttc tccgtctccg gcgagggcga gggcgacgcc acctacggca agctcaccct
     4141 caagttcatc tgcaccaccg gcaagctccc cgtcccctgg cccaccctcg tcaccacctt
     4201 cggctacggc ctcatgtgct tcgcccgtta ccccgaccac atgaagcagc acgacttctt
     4261 caagtccgcc atgcccgagg gctacgtcca ggagcgtacc atcttcttca aggacgacgg
     4321 caactacaag acccgtgccg aggtcaagtt cgagggcgac accctcgtca accgtatcga
     4381 gctcaagggc atcgacttca aggaggacgg caacatcctc ggccacaagc tcgagtacaa
     4441 ctacaactcc cacaacgtct acatcatggc cgacaagcag aagaacggca tcaaggtcaa
     4501 cttcaagatc cgtcacaaca tcgaggacgg ctccgtccag ctcgccgacc actaccagca
     4561 gaacaccccc atcggcgacg gccccgtcct cctccccgac aaccactacc tctcctacca
     4621 gtccgccctc tccaaggacc ccaacgagaa gcgtgaccac atggtcctcc tcgagttcgt
     4681 caccgccgcc ggcatcactc tcggcatgga cgagctttac aagtagaagg ttgagcgcgt
     4741 aatagtgggt tggtcttgtc tctatccatt catctgtgag acatactgat ttcaactttt
     4801 tcatcagata ataaggatgg tgtgagataa gcaaggctgg gttatgaggt ttgggactag
     4861 atggtgtttc gcgtctaaag gtgagtcttc gttggttcaa tcacattgat ctgatcgctg
     4921 atcaattgtc agcctctgaa tgtgctcttg aagaaaatgg ttgtctgtaa gaatccagaa
     4981 ccaaaaacgg tcatttcaag tatgtatcct ccgtgaagtt cctttagctc ttgctaatat
     5041 tggactttag cgtgctttag ccaaagacag ggtcgcgagg ttaagaggat gggccaggaa
     5101 aaatctaagt tagatttgtc acacaaagaa aaagattgtc tcagttttgg ttttgtttgg
     5161 tttgatttat gcataggaaa tatttatgat cgaaatcact catttcctgc ttcgtaaata
     5221 tcatctacta tatgcttcgt aaatgtccat ctcctacata ttgtggatat gataaatgca
     5281 atcctctcgt ctacactctc ggcttatgcc tttgtcttct ttgcgctcct tttagccttc
     5341 tttttattcc tagtcttcaa gcccatcact agttctagag cggccgccac cgcggtggag
     5401 ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
     5461 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5521 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5581 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5641 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5701 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5761 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5821 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5881 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5941 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6001 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6061 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6121 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6181 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6241 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6301 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6361 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6421 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6481 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6541 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6601 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6661 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6721 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6781 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6841 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6901 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6961 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7021 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7081 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7141 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7201 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7261 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7321 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7381 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7441 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7501 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7561 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag
     7621 cggcaagatc tgcccataga cttcaaatca tggaattgga cgtttatctt cttgctttgg
     7681 atatccaccc tatcgtacaa tcttggcact ttggatcata cgaggcactt tgcggactag
     7741 ggaatggtta tcatcaattt cttggacctc tccatgacac ctggcatcgt cgagctacca
     7801 cagccccctg aattcttaaa ggcgaacccc ccttcctagg aaccttgagc gctggactgg
     7861 atggcactct gagaccgcct atgcttaagg acgcgagctt acccttccat tgatggaagt
     7921 cgatcattta tgataataga acacggcgga acttgacttt acaggccaag ggaagcaaca
     7981 ggagaagaag ctaaggttat aacaggctca gcatatttgt atatacggtg ccattcaacg
     8041 ctgaacactt tgtactcacg gtctgcccgt tgaataacac catctgctga tgatcatccc
     8101 aatttgtgag tcttatttgc cattgatcct ggtgtataac cgatactgaa gccctttagg
     8161 tctgatcagt tatagacgtg gtatattcat tcttcctaga ttagctatgc cgaaagagat
     8221 ggataccttg atgtgactgg aatgccatga agcaggagat tgtagcagct caaattggag
     8281 aaggatgttg caaccgtcgg ccgtgaaggt cttgtcatat ctttgtctgc ctgggagaat
     8341 atgtacaagt gccccgatgt tactgatgga aaacatcgtg tggcgcgcca cgatacttgt
     8401 gttaattaa
//
