LOCUS       sequence-292550-        8412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluoroescent marker for use in Cryptococcus neoformans. Contains a 
            non-codon optimised GFP S65T marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8412)
  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 8412)
  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: pGWKS2
FEATURES             Location/Qualifiers
     source          1..8412
                     /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"
     CDS             join(4007..4009,4010..4012,4013..4726)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(4052..4073)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4313..4332)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4660..4681
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5419..5439)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5419..5437)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5458..5474)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5458..5474)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5471..5493)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5482..5498
                     /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(5506..5512,5513..5530,5531..5536))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5551..5572
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5689..5706)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5860..6448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5940..5959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6619..7410,7411..7479))
                     /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     7242..7261
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7480..7584)
                     /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 tgagcaaggg
     4021 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtgaacgg
     4081 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     4141 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt
     4201 cacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     4261 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     4321 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     4381 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     4441 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     4501 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     4561 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     4621 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     4681 gaccgccgcc gggatcactc acggcatgga cgagctgtac aagtaataga aggttgagcg
     4741 cgtaatagtg ggttggtctt gtctctatcc attcatctgt gagacatact gatttcaact
     4801 ttttcatcag ataataagga tggtgtgaga taagcaaggc tgggttatga ggtttgggac
     4861 tagatggtgt ttcgcgtcta aaggtgagtc ttcgttggtt caatcacatt gatctgatcg
     4921 ctgatcaatt gtcagcctct gaatgtgctc ttgaagaaaa tggttgtctg taagaatcca
     4981 gaaccaaaaa cggtcatttc aagtatgtat cctccgtgaa gttcctttag ctcttgctaa
     5041 tattggactt tagcgtgctt tagccaaaga cagggtcgcg aggttaagag gatgggccag
     5101 gaaaaatcta agttagattt gtcacacaaa gaaaaagatt gtctcagttt tggttttgtt
     5161 tggtttgatt tatgcatagg aaatatttat gatcgaaatc actcatttcc tgcttcgtaa
     5221 atatcatcta ctatatgctt cgtaaatgtc catctcctac atattgtgga tatgataaat
     5281 gcaatcctct cgtctacact ctcggcttat gcctttgtct tctttgcgct ccttttagcc
     5341 ttctttttat tcctagtctt caagcccatc actagttcta gagcggccgc caccgcggtg
     5401 gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     5461 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5521 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5581 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5641 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5701 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5761 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5821 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5881 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5941 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6001 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6061 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6121 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6181 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6241 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6301 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6361 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6421 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6481 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6541 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6601 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6661 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6721 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6781 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6841 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6901 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6961 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7021 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7081 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7141 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7201 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7261 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7321 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7381 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7441 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7501 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7561 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg
     7621 tagcggcaag atctgcccat agacttcaaa tcatggaatt ggacgtttat cttcttgctt
     7681 tggatatcca ccctatcgta caatcttggc actttggatc atacgaggca ctttgcggac
     7741 tagggaatgg ttatcatcaa tttcttggac ctctccatga cacctggcat cgtcgagcta
     7801 ccacagcccc ctgaattctt aaaggcgaac cccccttcct aggaaccttg agcgctggac
     7861 tggatggcac tctgagaccg cctatgctta aggacgcgag cttacccttc cattgatgga
     7921 agtcgatcat ttatgataat agaacacggc ggaacttgac tttacaggcc aagggaagca
     7981 acaggagaag aagctaaggt tataacaggc tcagcatatt tgtatatacg gtgccattca
     8041 acgctgaaca ctttgtactc acggtctgcc cgttgaataa caccatctgc tgatgatcat
     8101 cccaatttgt gagtcttatt tgccattgat cctggtgtat aaccgatact gaagcccttt
     8161 aggtctgatc agttatagac gtggtatatt cattcttcct agattagcta tgccgaaaga
     8221 gatggatacc ttgatgtgac tggaatgcca tgaagcagga gattgtagca gctcaaattg
     8281 gagaaggatg ttgcaaccgt cggccgtgaa ggtcttgtca tatctttgtc tgcctgggag
     8341 aatatgtaca agtgccccga tgttactgat ggaaaacatc gtgtggcgcg ccacgatact
     8401 tgtgttaatt aa
//
