LOCUS Exported 8400 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Fluoroescent marker for use in Cryptococcus neoformans. Contains a non-codon optimised mCherry marker.. ACCESSION . VERSION . KEYWORDS pGWKS17 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8400 /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" 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(5494..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(6607..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 //