LOCUS Exported 6628 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid for tagging proteins in Cryptococcus neoformans; mCherry tag, Neo selection. ACCESSION . VERSION . KEYWORDS YSBE 524 : pNEO_mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6628) 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 6628) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6628 /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(467..1261) /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 GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" CDS complement(2384..3091) /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(2662..2681) /label=mCherry-F /note="mCherry, forward primer" primer_bind 2931..2949 /label=mCherry-R /note="mCherry, reverse primer" primer_bind complement(3211..3228) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3211..3227) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3220..3242) /label=M13/pUC Forward /note="In lacZ gene" CDS 3365..3667 /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 3546..3565 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 4016..4810 /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" CDS 4828..5688 /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(5046..5065) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5859..6447 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6348..6367 /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 gtaccgagct cggatccgga gccatgaaga tcctgaggat tcaacggcgt tgctgagggt 301 gggaaggatg gtattgttgt ctgtagatag attaatgggc atataatgtc aaaaaagatg 361 attgacacga tgtctgtctc gaattcgcag tgtgctaaaa ttgtatcact tctctaatat 421 gtatatatac accctctaag gaaaattaac cccttaccgc cttcactcag aagaactcgt 481 caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga 541 ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta 601 tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc 661 cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc 721 cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct 781 cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga 841 tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc 901 gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgggat gacaggagat 961 cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga 1021 gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct 1081 gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg 1141 ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc 1201 cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca 1261 tagacatgtt gggcgagttt actaatggaa aaagacaatt agcaaagagg ctgcatatta 1321 ggagacgcgg tgaagattgg ggaggacaat agggtccgtg caccaaaaag tcctgacggc 1381 agctctgttc aacgcacatc catctcttcc agctatgcgt gtggcaccag cacggatgga 1441 ttgttgcacc aaaaggacaa ggcgagatgg atgcaggtcg aggcgatgcg gtgctatcgg 1501 ccctcgttgt cgaacgccac gtcgtcctct cggcgaccgt caatcctgga aggaaatgac 1561 caagcgtgca cccagcatcc agcatccaac atccagcacc cagcacctag cacccatgat 1621 ccaatggccg gcatgcagat cagcgtgggg agagcaccat ctagcagccc ctggcaggcc 1681 cagcattgtg ctcggatgtg cgataggcgg tgggtgcgat aaggtggatt agtggaggat 1741 ggaggactga gtggcgggca agggaagggg gcgggaagat gactcacctt gggcagtggg 1801 tgggggatgc ggtatagggg atgtatgtgg atgctggcgg aggatagaag ctgtaagctg 1861 agctggtgcc aacggtgacg ctgtgagagt ggtctcggcg accctgacgg cgactcggcg 1921 ttaccccgtg tggcacaccg gacgcgcgtc gagtgacgtt gctgctattg ttgttaccat 1981 catcctctcc tccgacaacc atacactcag tgcacaccat gccactcgaa tcctgcatgc 2041 ggatccacta gtaacggccg ccagtgtgct ggaattcgcc cttgtaccaa tctatccctc 2101 tctccgaaca atctccacat tccctccctt ctgctccctg aacccgcaac acgagccgtc 2161 cacacttctt ctcaactttg cgcttctcaa cttttgctta tgcaatcttg ctccattatc 2221 tatcatccca acttatcccg actcttcctc gtcccaccgc ccaccacaca ccacattact 2281 ttcaggctcc tgaatacaac acatcattca taagttcaag tatacgccaa catctcttct 2341 ttcgttataa ataaaaagaa agaaagaaag aagaacgatt cttttacttg tacagctcgt 2401 ccatgccgcc ggtggagtgg cggccctcgg cgcgttcgta ctgttccacg atggtgtagt 2461 cctcgttgtg ggaggtgatg tccaacttga tgttgacgtt gtaggcgccg ggcagcggca 2521 cgggcttctt ggccttgtag gtggtcttga cctcagcgtc gtagtggccg ccgtccttca 2581 gcttcagcct ctgcttgatc tcgcccttca gggcgccgtc ctcggggtac atccgctcgg 2641 aggaggcctc ccagcccatg gtcttcttct gcattacggg gccgtcggag gggaagttgg 2701 tgccgcgcag cctcaccttg tagatgaact cgccgtcctg cagggaggag tcctgggtca 2761 cggtcaccac gccgccgtcc tcgaagttca tcacgcgctc ccacttgaag ccctcgggga 2821 aggacagctt caagtagtcg gggatgtcgg cggggtgctt cacgtaggcc ttggagccgt 2881 acatgaactg aggggacagg atgtcccagg cgaagggcag ggggccaccc ttggtcacct 2941 tcagcttggc ggtctgggtg ccctcgtagg ggcggccctc gccctcgccc tcgatctcga 3001 actcgtggcc gttcacggag ccctccatgt gcaccttgaa gcgcatgaac tccttgatga 3061 tggccatgtt atcctcctcg cccttgctca cagagccacc gccacctgcg gccgcaaggg 3121 cgaattctgc agatatccat cacactggcg gccgctcgag catgcatcta gagggcccaa 3181 ttcgccctat agtgaatcgt attacaattc actggccgtc gttttacaac gtcgtgactg 3241 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 3301 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctatacgt 3361 acggcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt 3421 acagagtgat attattgaca cgccggggcg acggatggtg atccccctgg ccagtgcacg 3481 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag 3541 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt 3601 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg 3661 aatataaatg tcaggcatga gattatcaaa aaggatcttc acctagatcc ttttcacgta 3721 gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg 3781 gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg 3841 atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc 3901 ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat 3961 ctgatggcgc aggggatcaa gctctgatca agagacagga tgaggatcgt ttcgcatgat 4021 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta 4081 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca 4141 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga 4201 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga 4261 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct 4321 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg 4381 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga 4441 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca 4501 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga 4561 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg 4621 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc 4681 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt 4741 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga 4801 gttcttctga attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 4861 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 4921 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 4981 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 5041 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 5101 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 5161 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 5221 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 5281 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 5341 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 5401 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 5461 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 5521 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 5581 aagccctccc gtatcgtagt tatctacacg acggggagcc aggcaactat ggatgaacga 5641 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 5701 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 5761 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 5821 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 5881 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 5941 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 6001 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 6061 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 6121 cttaccgggt tggattcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 6181 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 6241 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 6301 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 6361 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 6421 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 6481 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 6541 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 6601 agcgaatcag tgagcgagga agcggaag //