LOCUS       Exported                8403 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Fluoroescent marker for use in Cryptococcus neoformans. Contains a 
            codon optimised mRuby3 marker..
ACCESSION   .
VERSION     .
KEYWORDS    pGWKS7
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8403)
  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 8403)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8403
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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(5410..5430)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5410..5428)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5449..5465)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5449..5465)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5462..5484)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5473..5489
                     /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(5497..5527)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5542..5563
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5680..5697)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5851..6439)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5931..5950)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6610..7470)
                     /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     7233..7252
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7471..7575)
                     /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 atcaaggaga acatgcgtat gaaggtcgtc atggagggtt ctgtcaacgg
     4081 ccaccagttc aagtgcactg gtgagggtga gggcaggccc tacgagggtg tccagaccat
     4141 gaggatcaag gtcatcgagg gtggtcccct ccctttcgcc ttcgacattc ttgccacctc
     4201 tttcatgtac ggctcccgta ccttcatcaa gtaccccgcc gacatccctg acttcttcaa
     4261 gcagtccttc cctgagggtt tcacttggga gagggttacc aggtacgagg acggtggtgt
     4321 cgtcaccgtc acccaggaca cctcccttga ggacggcgag ctcgtctaca acgtcaaggt
     4381 caggggcgtt aacttcccct ccaacggtcc cgtcatgcag aagaagacca agggttggga
     4441 gcctaacact gagatgatgt accctgctga cggtggtctc aggggttaca ctgacatcgc
     4501 tctcaaggtt gacggtggtg gccacctcca ctgcaacttc gtcactactt acaggtctaa
     4561 gaagaccgtc ggcaacatca agatgcccgg tgtccacgcc gttgaccacc gtctcgagag
     4621 gatcgaggag tccgacaacg agacctacgt tgtccagagg gaggtcgctg ttgccaagta
     4681 ctccaacctt ggtggtggca tggacgagct ctacaagtag aaggttgagc gcgtaatagt
     4741 gggttggtct tgtctctatc cattcatctg tgagacatac tgatttcaac tttttcatca
     4801 gataataagg atggtgtgag ataagcaagg ctgggttatg aggtttggga ctagatggtg
     4861 tttcgcgtct aaaggtgagt cttcgttggt tcaatcacat tgatctgatc gctgatcaat
     4921 tgtcagcctc tgaatgtgct cttgaagaaa atggttgtct gtaagaatcc agaaccaaaa
     4981 acggtcattt caagtatgta tcctccgtga agttccttta gctcttgcta atattggact
     5041 ttagcgtgct ttagccaaag acagggtcgc gaggttaaga ggatgggcca ggaaaaatct
     5101 aagttagatt tgtcacacaa agaaaaagat tgtctcagtt ttggttttgt ttggtttgat
     5161 ttatgcatag gaaatattta tgatcgaaat cactcatttc ctgcttcgta aatatcatct
     5221 actatatgct tcgtaaatgt ccatctccta catattgtgg atatgataaa tgcaatcctc
     5281 tcgtctacac tctcggctta tgcctttgtc ttctttgcgc tccttttagc cttcttttta
     5341 ttcctagtct tcaagcccat cactagttct agagcggccg ccaccgcggt ggagctccag
     5401 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     5461 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5521 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5581 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5641 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5701 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5761 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5821 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5881 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5941 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6001 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6061 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6121 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6181 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6241 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6301 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6361 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6421 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6481 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6541 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6601 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6661 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6721 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6781 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6841 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6901 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6961 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7021 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7081 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7141 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7201 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7261 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7321 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7381 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7441 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7501 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7561 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcaa
     7621 gatctgccca tagacttcaa atcatggaat tggacgttta tcttcttgct ttggatatcc
     7681 accctatcgt acaatcttgg cactttggat catacgaggc actttgcgga ctagggaatg
     7741 gttatcatca atttcttgga cctctccatg acacctggca tcgtcgagct accacagccc
     7801 cctgaattct taaaggcgaa ccccccttcc taggaacctt gagcgctgga ctggatggca
     7861 ctctgagacc gcctatgctt aaggacgcga gcttaccctt ccattgatgg aagtcgatca
     7921 tttatgataa tagaacacgg cggaacttga ctttacaggc caagggaagc aacaggagaa
     7981 gaagctaagg ttataacagg ctcagcatat ttgtatatac ggtgccattc aacgctgaac
     8041 actttgtact cacggtctgc ccgttgaata acaccatctg ctgatgatca tcccaatttg
     8101 tgagtcttat ttgccattga tcctggtgta taaccgatac tgaagccctt taggtctgat
     8161 cagttataga cgtggtatat tcattcttcc tagattagct atgccgaaag agatggatac
     8221 cttgatgtga ctggaatgcc atgaagcagg agattgtagc agctcaaatt ggagaaggat
     8281 gttgcaaccg tcggccgtga aggtcttgtc atatctttgt ctgcctggga gaatatgtac
     8341 aagtgccccg atgttactga tggaaaacat cgtgtggcgc gccacgatac ttgtgttaat
     8401 taa
//
