LOCUS Exported 6415 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Plasmid for tagging proteins in Cryptococcus neoformans; mCherry tag, NAt selection. ACCESSION . VERSION . KEYWORDS YSBE 596 : pNAT_mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6415) 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 6415) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6415 /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 1031..1594 /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..2878) /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVRLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVPLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(2449..2468) /label=mCherry-F /note="mCherry, forward primer" primer_bind 2718..2736 /label=mCherry-R /note="mCherry, reverse primer" primer_bind complement(2998..3015) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2998..3014) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3007..3029) /label=M13/pUC Forward /note="In lacZ gene" CDS 3152..3454 /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 3333..3352 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 3803..4597 /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(3857..3876) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4467..4486 /label=Neo-F /note="Neomycin resistance gene, forward primer" CDS 4615..5475 /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(4833..4852) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5646..6234 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6135..6154 /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 catgcaggat tcgagtggca tggtgtgcgc tgagtgtatg gttgtcggag gagaggatga 301 tggtaacaac aatagcagca acgtcactcg acgcgcgtcc ggtgtgccac acggggtaac 361 gccgagtcgc cgtcagggtc gccgagacca ctctcacagc gtcaccgttg gcaccagctc 421 agcttacagc ttctatcctc cgccagcatc cacatacatc ccctataccg catcccccac 481 ccactgccca aggtgagtca tcttcccgcc cccttccctt gcccgccact cagtcctcca 541 tcctccacta atccacctta tcgcacccac cgcctatcgc acatccgagc acaatgctgg 601 gcctgccagg ggctgctaga tggtgctctc cccacgctga tctgcatgcc ggccattgga 661 tcatgggtgc taggtgctgg gtgctggatg ttggatgctg gatgctgggt gcacgcttgg 721 tcatttcctt ccaggattga cggtcgccga gaggacgacg tggcgttcga caacgagggc 781 cgatagcacc gcatcgcctc gacctgcatc catctcgcct tgtccttttg gtgcaacaat 841 ccatccgtgc tggtgccaca cgcatagctg gaagagatgg atgtgcgttg aacagagctg 901 ccgtcaggac tttttggtgc acggacccta ttgtcctccc caatcttcac cgcgtctcct 961 aatatgcagc ctctttgcta attgtctttt tccattagta aactcgccca acatgtctat 1021 ggcggccgcc actcttgacg acacggctta ccggtaccgc accagtgtcc cgggggacgc 1081 cgaggccatc gaggcactgg atgggtcctt caccaccgac accgtcttcc gcgtcaccgc 1141 caccggggac ggcttcaccc tgcgggaggt gccggtggac ccgcccctga ccaaggtgtt 1201 ccccgacgac gaatcggacg acgaatcgga cgacggggag gacggcgacc cggactcccg 1261 gacgttcgtc gcgtacgggg acgacggcga cctggcgggc ttcgtggtcg tctcgtactc 1321 cggctggaac cgccggctga ccgtcgagga catcgaggtc gccccggagc accgggggca 1381 cggggtcggg cgcgcgttga tggggctcgc gacggagttc gcccgcgagc ggggcgccgg 1441 gcacctctgg ctggaggtca ccaacgtcaa cgcaccggcg atccacgcgt accggcggat 1501 ggggttcacc ctctgcggcc tggacaccgc cctgtacgac ggcaccgcct cggacggcga 1561 gcaggcgctc tacatgagca tgccctgccc ctaagaattc gtgaaggcgg taaggggtta 1621 attttcctta gagggtgtat atatacatat tagagaagtg atacaatttt agcacactgc 1681 gaatccgaga cagacatcgt gtcaatcatc ttttttgaca ttatatgccc attaatctat 1741 ctacagacaa caataccatc cttcccaccc tcagcaacgc cgttgaatcc tcaggatctt 1801 catggctcca agcttggtac cgagctcgga tccactagta acggccgcca gtgtgctgga 1861 attcgccctt gtaccaatct atccctctct ccgaacaatc tccacattcc ctcccttctg 1921 ctccctgaac ccgcaacacg agccgtccac acttcttctc aactttgcgc ttctcaactt 1981 ttgcttatgc aatcttgctc cattatctat catcccaact tatcccgact cttcctcgtc 2041 ccaccgccca ccacacacca cattactttc aggctcctga atacaacaca tcattcataa 2101 gttcaagtat acgccaacat ctcttctttc gttataaata aaaagaaaga aagaaagaag 2161 aacgattctt ttacttgtac agctcgtcca tgccgccggt ggagtggcgg ccctcggcgc 2221 gttcgtactg ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc aacttgatgt 2281 tgacgttgta ggcgccgggc agcggcacgg gcttcttggc cttgtaggtg gtcttgacct 2341 cagcgtcgta gtggccgccg tccttcagct tcagcctctg cttgatctcg cccttcaggg 2401 cgccgtcctc ggggtacatc cgctcggagg aggcctccca gcccatggtc ttcttctgca 2461 ttacggggcc gtcggagggg aagttggtgc cgcgcagcct caccttgtag atgaactcgc 2521 cgtcctgcag ggaggagtcc tgggtcacgg tcaccacgcc gccgtcctcg aagttcatca 2581 cgcgctccca cttgaagccc tcggggaagg acagcttcaa gtagtcgggg atgtcggcgg 2641 ggtgcttcac gtaggccttg gagccgtaca tgaactgagg ggacaggatg tcccaggcga 2701 agggcagggg gccacccttg gtcaccttca gcttggcggt ctgggtgccc tcgtaggggc 2761 ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc tccatgtgca 2821 ccttgaagcg catgaactcc ttgatgatgg ccatgttatc ctcctcgccc ttgctcacag 2881 agccaccgcc acctgcggcc gcaagggcga attctgcaga tatccatcac actggcggcc 2941 gctcgagcat gcatctagag ggcccaattc gccctatagt gaatcgtatt acaattcact 3001 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 3061 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 3121 ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga 3181 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg 3241 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta 3301 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt 3361 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa 3421 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag 3481 gatcttcacc tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg 3541 gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca 3601 ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag 3661 cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa 3721 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga 3781 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc 3841 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 3901 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 3961 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac 4021 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 4081 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 4141 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 4201 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 4261 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 4321 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 4381 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 4441 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 4501 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 4561 catcgccttc tatcgccttc ttgacgagtt cttctgaatt gaaaaaggaa gagtatgagt 4621 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 4681 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 4741 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 4801 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 4861 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 4921 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 4981 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 5041 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 5101 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 5161 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 5221 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 5281 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 5341 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 5401 gggagccagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 5461 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 5521 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 5581 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 5641 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 5701 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 5761 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac 5821 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 5881 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg attcaagacg atagttaccg 5941 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6001 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6061 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 6121 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 6181 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 6241 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 6301 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 6361 gctcgccgca gccgaacgac cgagcgcagc gaatcagtga gcgaggaagc ggaag //