LOCUS       sequence-292450-        8400 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluoroescent marker for use in Cryptococcus neoformans. Contains a 
            non-codon optimised mCherry marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8400)
  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 8400)
  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: pGWKS17
FEATURES             Location/Qualifiers
     source          1..8400
                     /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             4007..4717
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4152..4170)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4420..4439
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(5407..5427)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5407..5425)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5446..5462)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5446..5462)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5459..5481)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5470..5486
                     /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(5494..5500,5501..5518,5519..5524))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5539..5560
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5677..5694)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5848..6436)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5928..5947)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6607..7398,7399..7467))
                     /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     7230..7249
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7468..7572)
                     /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 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     4081 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     4141 cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat
     4201 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     4261 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     4321 ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta
     4381 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     4441 catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     4501 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac
     4561 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga
     4621 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     4681 ccactccacc ggcggcatgg acgagctgta caagtagaag gttgagcgcg taatagtggg
     4741 ttggtcttgt ctctatccat tcatctgtga gacatactga tttcaacttt ttcatcagat
     4801 aataaggatg gtgtgagata agcaaggctg ggttatgagg tttgggacta gatggtgttt
     4861 cgcgtctaaa ggtgagtctt cgttggttca atcacattga tctgatcgct gatcaattgt
     4921 cagcctctga atgtgctctt gaagaaaatg gttgtctgta agaatccaga accaaaaacg
     4981 gtcatttcaa gtatgtatcc tccgtgaagt tcctttagct cttgctaata ttggacttta
     5041 gcgtgcttta gccaaagaca gggtcgcgag gttaagagga tgggccagga aaaatctaag
     5101 ttagatttgt cacacaaaga aaaagattgt ctcagttttg gttttgtttg gtttgattta
     5161 tgcataggaa atatttatga tcgaaatcac tcatttcctg cttcgtaaat atcatctact
     5221 atatgcttcg taaatgtcca tctcctacat attgtggata tgataaatgc aatcctctcg
     5281 tctacactct cggcttatgc ctttgtcttc tttgcgctcc ttttagcctt ctttttattc
     5341 ctagtcttca agcccatcac tagttctaga gcggccgcca ccgcggtgga gctccagctt
     5401 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     5461 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5521 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5581 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5641 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5701 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5761 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5821 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5881 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5941 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6001 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6061 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6121 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6181 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6241 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6301 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6361 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6421 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6481 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6541 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6601 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6661 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6721 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6781 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6841 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6901 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6961 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7021 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7081 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7141 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7201 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7261 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7321 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7381 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7441 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7501 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7561 aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta gcggcaagat
     7621 ctgcccatag acttcaaatc atggaattgg acgtttatct tcttgctttg gatatccacc
     7681 ctatcgtaca atcttggcac tttggatcat acgaggcact ttgcggacta gggaatggtt
     7741 atcatcaatt tcttggacct ctccatgaca cctggcatcg tcgagctacc acagccccct
     7801 gaattcttaa aggcgaaccc cccttcctag gaaccttgag cgctggactg gatggcactc
     7861 tgagaccgcc tatgcttaag gacgcgagct tacccttcca ttgatggaag tcgatcattt
     7921 atgataatag aacacggcgg aacttgactt tacaggccaa gggaagcaac aggagaagaa
     7981 gctaaggtta taacaggctc agcatatttg tatatacggt gccattcaac gctgaacact
     8041 ttgtactcac ggtctgcccg ttgaataaca ccatctgctg atgatcatcc caatttgtga
     8101 gtcttatttg ccattgatcc tggtgtataa ccgatactga agccctttag gtctgatcag
     8161 ttatagacgt ggtatattca ttcttcctag attagctatg ccgaaagaga tggatacctt
     8221 gatgtgactg gaatgccatg aagcaggaga ttgtagcagc tcaaattgga gaaggatgtt
     8281 gcaaccgtcg gccgtgaagg tcttgtcata tctttgtctg cctgggagaa tatgtacaag
     8341 tgccccgatg ttactgatgg aaaacatcgt gtggcgcgcc acgatacttg tgttaattaa
//
