LOCUS Exported 6118 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Plasmid for tagging proteins in Cryptococcus neoformans; 6xHA tag, Neo selection. ACCESSION . VERSION . KEYWORDS YSBE 517 : pNEO_6xHA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6118) 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 6118) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6118 /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 1019..1813 /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(2393..2482) /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" CDS complement(2492..2581) /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" primer_bind complement(2701..2718) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2701..2717) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2710..2732) /label=M13/pUC Forward /note="In lacZ gene" CDS 2855..3157 /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 3036..3055 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 3506..4300 /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 4318..5178 /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(4536..4555) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5349..5937 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5838..5857 /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 tggtgtgcac 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 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 1081 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 1141 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 1201 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 1261 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 1321 tctcctgtca tcccaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 1381 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 1441 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 1501 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 1561 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 1621 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 1681 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 1741 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 1801 cgagttcttc tgagtgaagg cggtaagggg ttaattttcc ttagagggtg tatatataca 1861 tattagagaa gtgatacaat tttagcacac tgcgaattcg agacagacat cgtgtcaatc 1921 atcttttttg acattatatg cccattaatc tatctacaga caacaatacc atccttccca 1981 ccctcagcaa cgccgttgaa tcctcaggat cttcatggct ccaagcttgg taccgagctc 2041 ggatccacta gtaacggccg ccagtgtgct ggaattcgcc cttgtaccaa tctatccctc 2101 tctccgaaca atctccacat tccctccctt ctgctccctg aacccgcaac acgagccgtc 2161 cacacttctt ctcaactttg cgcttctcaa cttttgctta tgcaatcttg ctccaccatc 2221 tatcatccca acttatcccg actcttcctc gtcccaccgc ccaccacaca ccacattact 2281 ttcaggctcc tgaatacaac acatcattca taagttcaag tatacgccaa catctcttct 2341 ttcgttataa ataaaaagaa agaaagaaag aagaacgatt ctttctagct taagcgtaat 2401 ctggaacgtc atatggatag gatcctgcat agtccgggac gtcataggga tagcccgcat 2461 agtcaggaac atcgtatggg tattctagac aagcgtaatc tggaacgtca tatggatagg 2521 atcctgcata gtccgggacg tcatagggat agcccgcata gtcaggaaca tcgtatgggt 2581 atggcgcgcc gcatgctgcg gccgcaaggg cgaattctgc agatatccat cacactggcg 2641 gccgctcgag catgcatcta gagggcccaa ttcgccctat agtgaatcgt attacaattc 2701 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 2761 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 2821 cccttcccaa cagttgcgca gcctatacgt acggcagttt aaggtttaca cctataaaag 2881 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgccggggcg 2941 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact 3001 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag 3061 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat 3121 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggcatga gattatcaaa 3181 aaggatcttc acctagatcc ttttcacgta gaaagccagt ccgcagaaac ggtgctgacc 3241 ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa 3301 gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc 3361 aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt 3421 aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca 3481 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 3541 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 3601 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 3661 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac 3721 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 3781 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 3841 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 3901 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 3961 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 4021 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 4081 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 4141 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 4201 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 4261 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attgaaaaag gaagagtatg 4321 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 4381 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 4441 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 4501 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 4561 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 4621 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 4681 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 4741 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 4801 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 4861 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 4921 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 4981 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 5041 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 5101 acggggagcc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 5161 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 5221 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 5281 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 5341 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 5401 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 5461 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc 5521 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 5581 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggattcaag acgatagtta 5641 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 5701 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 5761 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 5821 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 5881 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 5941 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 6001 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 6061 accgctcgcc gcagccgaac gaccgagcgc agcgaatcag tgagcgagga agcggaag //