LOCUS Exported 6424 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid for tagging proteins in Cryptococcus neoformans; GFP tag, NAT selection. ACCESSION . VERSION . KEYWORDS YSBE 557 : pNAT_GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6424) AUTHORS So YS, Yang DH, Jung KW, Huh WK, Bahn YS TITLE Molecular Characterization of Adenylyl Cyclase Complex Proteins Using Versatile Protein-Tagging Plasmid Systems in Cryptococcus neoformans. JOURNAL J Microbiol Biotechnol. 2017 Feb 28;27(2):357-364. doi: 10.4014/jmb.1609.09036. PUBMED 27780958 REFERENCE 2 (bases 1 to 6424) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6424 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /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)." primer_bind 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS complement(455..1018) /codon_start=1 /gene="Streptomyces noursei nat1" /product="nourseothricin acetyltransferase" /label=NrsR /note="confers resistance to nourseothricin" /translation="TLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFTLR EVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRRLT VEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTLCG LDTALYDGTASDGEQALYMSMPCP" CDS complement(2171..2887) /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITHGMDELYK" primer_bind complement(2216..2237) /label=EGFP-C /note="EGFP, forward primer" primer_bind 2565..2584 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2824..2845 /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3007..3024) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3007..3023) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3016..3038) /label=M13/pUC Forward /note="In lacZ gene" CDS 3161..3463 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 3342..3361 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 3812..4606 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3866..3885) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4476..4495 /label=Neo-F /note="Neomycin resistance gene, forward primer" CDS 4624..5484 /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 complement(4842..4861) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5655..6243 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6144..6163 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg 241 gagccatgaa gatcctgagg attcaacggc gttgctgagg gtgggaagga tggtattgtt 301 gtctgtagat agattaatgg gcatataatg tcaaaaaaga tgattgacac gatgtctgtc 361 tcggattcgc agtgtgctaa aattgtatca cttctctaat atgtatatat acaccctcta 421 aggaaaatta accccttacc gccttcacga attcttaggg gcagggcatg ctcatgtaga 481 gcgcctgctc gccgtccgag gcggtgccgt cgtacagggc ggtgtccagg ccgcagaggg 541 tgaaccccat ccgccggtac gcgtggatcg ccggtgcgtt gacgttggtg acctccagcc 601 agaggtgccc ggcgccccgc tcgcgggcga actccgtcgc gagccccatc aacgcgcgcc 661 cgaccccgtg cccccggtgc tccggggcga cctcgatgtc ctcgacggtc agccggcggt 721 tccagccgga gtacgagacg accacgaagc ccgccaggtc gccgtcgtcc ccgtacgcga 781 cgaacgtccg ggagtccggg tcgccgtcct ccccgtcgtc cgattcgtcg tccgattcgt 841 cgtcggggaa caccttggtc aggggcgggt ccaccggcac ctcccgcagg gtgaagccgt 901 ccccggtggc ggtgacgcgg aagacggtgt cggtggtgaa ggacccatcc agtgcctcga 961 tggcctcggc gtcccccggg acactggtgc ggtaccggta agccgtgtcg tcaagagtgg 1021 cggccgccat agacatgttg ggcgagttta ctaatggaaa aagacaatta gcaaagaggc 1081 tgcatattag gagacgcggt gaagattggg gaggacaata gggtccgtgc accaaaaagt 1141 cctgacggca gctctgttca acgcacatcc atctcttcca gctatgcgtg tggcaccagc 1201 acggatggat tgttgcacca aaaggacaag gcgagatgga tgcaggtcga ggcgatgcgg 1261 tgctatcggc cctcgttgtc gaacgccacg tcgtcctctc ggcgaccgtc aatcctggaa 1321 ggaaatgacc aagcgtgcac ccagcatcca gcatccaaca tccagcaccc agcacctagc 1381 acccatgatc caatggccgg catgcagatc agcgtgggga gagcaccatc tagcagcccc 1441 tggcaggccc agcattgtgc tcggatgtgc gataggcggt gggtgcgata aggtggatta 1501 gtggaggatg gaggactgag tggcgggcaa gggaaggggg cgggaagatg actcaccttg 1561 ggcagtgggt gggggatgcg gtatagggga tgtatgtgga tgctggcgga ggatagaagc 1621 tgtaagctga gctggtgcca acggtgacgc tgtgagagtg gtctcggcga ccctgacggc 1681 gactcggcgt taccccgtgt ggcacaccgg acgcgcgtcg agtgacgttg ctgctattgt 1741 tgttaccatc atcctctcct ccgacaacca tacactcagc gcacaccatg ccactcgaat 1801 cctgcatgca agcttggtac cgagctcgga tccactagta acggccgcca gtgtgctgga 1861 attcgccctt gtaccaatct atccctctct ccgaacaatc tccacattcc ctcccttctg 1921 ctccctgaac ccgcaacacg agccgtccac acttcttctc aactttgcgc ttctcaactt 1981 ttgcttatgc aatcttgctc caccatctat catcccaact tatcccgact cttcctcgtc 2041 ccaccgccca ccacacacca cattactttc aggctcctga atacaacaca tcattcataa 2101 gttcaagtat acgccaacat ctcttctttc gttataaata aaaagaaaga aagaaagaag 2161 aacgattctt ttacttgtac agctcgtcca tgccgtgagt gatcccggcg gcggtcacga 2221 actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg 2281 tgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct 2341 ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca 2401 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt 2461 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga 2521 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt 2581 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga 2641 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtgaagg 2701 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg 2761 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt 2821 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct 2881 tgctcacaga gccaccgcca cctgcggccg caagggcgaa ttctgcagat atccatcaca 2941 ctggcggccg ctcgagcatg catctagagg gcccaattcg ccctatagtg aatcgtatta 3001 caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact 3061 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac 3121 cgatcgccct tcccaacagt tgcgcagcct atacgtacgg cagtttaagg tttacaccta 3181 taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc 3241 ggggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg 3301 tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat 3361 ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa 3421 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gcatgagatt 3481 atcaaaaagg atcttcacct agatcctttt cacgtagaaa gccagtccgc agaaacggtg 3541 ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa 3601 gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg 3661 gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg 3721 caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagctc 3781 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg 3841 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 3901 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 3961 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct 4021 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 4081 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 4141 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 4201 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 4261 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 4321 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 4381 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 4441 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 4501 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 4561 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgaattg aaaaaggaag 4621 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 4681 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 4741 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 4801 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 4861 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 4921 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 4981 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 5041 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 5101 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 5161 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 5221 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 5281 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 5341 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 5401 tacacgacgg ggagccaggc aactatggat gaacgaaata gacagatcgc tgagataggt 5461 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 5521 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 5581 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 5641 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 5701 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 5761 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 5821 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 5881 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ttcaagacga 5941 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 6001 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 6061 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 6121 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 6181 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 6241 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 6301 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 6361 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg aatcagtgag cgaggaagcg 6421 gaag //