LOCUS Exported 5827 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid for tagging proteins in Cryptococcus neoformans; 4xFLAG tag, NAT selection. ACCESSION . VERSION . KEYWORDS YSBE 558 : pNAT_4xFLAG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5827) 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 5827) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5827 /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(2180..2203) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS complement(2213..2236) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS complement(2240..2263) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS complement(2267..2290) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" primer_bind complement(2410..2427) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2410..2426) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2419..2441) /label=M13/pUC Forward /note="In lacZ gene" CDS 2564..2866 /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 2745..2764 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 3215..4009 /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(3269..3288) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3879..3898 /label=Neo-F /note="Neomycin resistance gene, forward primer" CDS 4027..4887 /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(4245..4264) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5058..5646 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5547..5566 /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 ttagtcgacc ttatcgtcgt catccttgta atcttctaga cacttgtcgt 2221 cgtcgtcctt gtagtctccc ttatcgtcat cgtctttgta gtcaccctta tcgtcgtcat 2281 ccttgtaatc tggcgcgccg catgctgcgg ccgcaagggc gaattctgca gatatccatc 2341 acactggcgg ccgctcgagc atgcatctag agggcccaat tcgccctata gtgaatcgta 2401 ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca 2461 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 2521 caccgatcgc ccttcccaac agttgcgcag cctatacgta cggcagttta aggtttacac 2581 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac 2641 gccggggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc 2701 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga 2761 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga 2821 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggcatgag 2881 attatcaaaa aggatcttca cctagatcct tttcacgtag aaagccagtc cgcagaaacg 2941 gtgctgaccc cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc 3001 aaagagaaag caggtagctt gcagtgggct tacatggcga tagctagact gggcggtttt 3061 atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc 3121 ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag 3181 ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc 3241 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat 3301 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt 3361 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg 3421 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag 3481 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc 3541 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc 3601 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga 3661 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga 3721 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg 3781 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg 3841 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc 3901 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc 3961 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttgaaaaagg 4021 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 4081 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 4141 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 4201 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 4261 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 4321 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 4381 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 4441 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 4501 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 4561 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 4621 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 4681 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 4741 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 4801 atctacacga cggggagcca ggcaactatg gatgaacgaa atagacagat cgctgagata 4861 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 4921 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 4981 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 5041 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 5101 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 5161 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 5221 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 5281 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggattcaaga 5341 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 5401 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 5461 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 5521 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 5581 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 5641 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 5701 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 5761 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgaatcagt gagcgaggaa 5821 gcggaag //