LOCUS Exported 8412 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Fluoroescent marker for use in Cryptococcus neoformans. Contains a non-codon optimised GFP S65T marker.. ACCESSION . VERSION . KEYWORDS pGWKS2 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8412 /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..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(5506..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(6619..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 //