LOCUS Exported 6859 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid for tagging proteins in Cryptococcus neoformans; mCherry tag, HYG selection. ACCESSION . VERSION . KEYWORDS YSBE 628 : pHYG_mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6859) 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 6859) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6859 /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(449..1474) /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" CDS complement(2616..3323) /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(2894..2913) /label=mCherry-F /note="mCherry, forward primer" primer_bind 3163..3181 /label=mCherry-R /note="mCherry, reverse primer" primer_bind complement(3442..3459) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3442..3458) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3451..3473) /label=M13/pUC Forward /note="In lacZ gene" CDS 3596..3898 /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 3777..3796 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 4247..5041 /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(4301..4320) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4911..4930 /label=Neo-F /note="Neomycin resistance gene, forward primer" CDS 5059..5919 /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(5277..5296) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6090..6678 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6579..6598 /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 tcgaattcgc agtgtgctaa aattgtatca cttctctaat atgtatatat acaccctcta 421 aggaaaatta accccttacc gccttcacct attcctttgc cctcggacga gtgctggggc 481 gtcggtttcc actatcggcg agtacttcta cacagccatc ggtccagacg gccgcgcttc 541 tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc 601 gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac caagctctga tagagttggt 661 caagaccaat gcggagcata tacgcccgga gccgcggcga tcctgcaagc tccggatgcc 721 tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc cagaagaaga 781 tgttggcgac ctcgtattgg gaatccccga acatcgcctc gctccagtca atgaccgctg 841 ttatgcggcc attgtccgtc aggacattgt tggagccgaa atccgcgtgc acgaggtgcc 901 ggacttcggg gcagtcctcg gcccaaagca tcagctcatc gagagcctgc gcgacggacg 961 cactgacggt gtcgtccatc acagtttgcc agtgatacac atggggatca gcaatcgcgc 1021 atatgaaatc acgccatgta gtgtattgac cgattccttg cggtccgaat gggccgaacc 1081 cgctcgtctg gctaagatcg gccgcagcga tcgcatccat ggcctccgcg accggctgca 1141 gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg 1201 agatgcaata ggtcaggctc tcgctgaatt ccccaatgtc aagcacttcc ggaatcggga 1261 gcgcggccga tgcaaagtgc cgataaacat aacgatcttt gtagaaacca tcggcgcagc 1321 tatttacccg caggacatat ccacgccctc ctacatcgaa gctgaaagca cgagattctt 1381 cgccctccga gagctgcatc aggtcggaga cgctgtcgaa cttttcgatc agaaacttct 1441 cgacagacgt cgcggtgagt tcaggctttt tcatagacat gttgggcgag tttactaatg 1501 gaaaaagaca attagcaaag aggctgcata ttaggagacg cggtgaagat tggggaggac 1561 aatagggtcc gtgcaccaaa aagtcctgac ggcagctctg ttcaacgcac atccatctct 1621 tccagctatg cgtgtggcac cagcacggat ggattgttgc accaaaagga caaggcgaga 1681 tggatgcagg tcgaggcgat gcggtgctat cggccctcgt tgtcgaacgc cacgtcgtcc 1741 tctcggcgac cgtcaatcct ggaaggaaat gaccaagcgt gcacccagca tccagcatcc 1801 aacatccagc acccagcacc tagcacccat gatccaatgg ccggcatgca gatcagcgtg 1861 gggagagcac catctagcag cccctggcag gcccagcatt gtgctcggat gtgcgatagg 1921 cggtgggtgc gataaggtgg attagtggag gatggaggac tgagtggcgg gcaagggaag 1981 ggggcgggaa gatgactcac cttgggcagt gggtggggga tgcggtatag gggatgtatg 2041 tggatgctgg cggaggatag aagctgtaag ctgagctggt gccaacggtg acgctgtgag 2101 agtggtctcg gcgaccctga cggcgactcg gcgttacccc gtgtggcaca ccggacgcgc 2161 gtcgagtgac gttgctgcta ttgttgttac catcatcctc tcctccgaca accatacact 2221 cagtgcacac catgccactc gaatcctgca tgcaagcttg gtaccgagct cggatccact 2281 agtaacggcc gccagtgtgc tggaattcgc ccttagtacc aatctatccc tctctccgaa 2341 caatctccac attccctccc ttctgctccc tgaacccgca acacgagccg tccacacttc 2401 ttctcaactt tgcgcttctc aacttttgct tatgcaatct tgctccacca tctatcatcc 2461 caacttatcc cgactcttcc tcgtcccacc gcccaccaca caccacatta ctttcaggct 2521 cctgaataca acacatcatt cataagttca agtatacgcc aacatctctt ctttcgttat 2581 aaataaaaag aaagaaagaa agaagaacga ttcttttact tgtacagctc gtccatgccg 2641 ccggtggagt ggcggccctc ggcgcgttcg tactgttcca cgatggtgta gtcctcgttg 2701 tgggaggtga tgtccaactt gatgttgacg ttgtaggcgc cgggcagctg cacgggcttc 2761 ttggccttgt aggtggtctt gacctcagcg tcgtagtggc cgccgtcctt cagcttcagc 2821 ctctgcttga tctcgccctt cagggcgccg tcctcggggt acatccgctc ggaggaggcc 2881 tcccagccca tggtcttctt ctgcattacg gggccgtcgg aggggaagtt ggtgccgcgc 2941 agcttcacct tgtagatgaa ctcgccgtcc tgcagggagg agtcctgggt cacggtcacc 3001 acgccgccgt cctcgaagtt catcacgcgc tcccacttga agccctcggg gaaggacagc 3061 ttcaagtagt cggggatgtc ggcggggtgc ttcacgtagg ccttggagcc gtacatgaac 3121 tgaggggaca ggatgtccca ggcgaagggc agggggccac ccttggtcac cttcagcttg 3181 gcggtctggg tgccctcgta ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg 3241 ccgttcacgg agccctccat gtgcaccttg aagcgcatga actccttgat gatggccatg 3301 ttatcctcct cgcccttgct cacagagcca ccgccacctg cggccgcagg gcgaattctg 3361 cagatatcca tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta 3421 tagtgaatcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 3481 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 3541 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt 3601 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga 3661 tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc 3721 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat 3781 gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct 3841 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat 3901 gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag 3961 tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga 4021 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga 4081 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa 4141 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg 4201 caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 4261 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 4321 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 4381 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc 4441 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 4501 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 4561 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 4621 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 4681 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 4741 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt 4801 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 4861 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 4921 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 4981 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 5041 aattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 5101 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 5161 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 5221 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 5281 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 5341 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 5401 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 5461 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 5521 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 5581 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 5641 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 5701 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 5761 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 5821 cgtatcgtag ttatctacac gacggggagc caggcaacta tggatgaacg aaatagacag 5881 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 5941 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 6001 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 6061 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 6121 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 6181 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt 6241 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 6301 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 6361 ttggattcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 6421 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 6481 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 6541 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 6601 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 6661 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 6721 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 6781 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgaatca 6841 gtgagcgagg aagcggaag //