LOCUS       sequence-177007-        5905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for tagging proteins in Cryptococcus neoformans; 6xHA tag, 
            NAT selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5905)
  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 5905)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.4014/jmb.1609"; 
            journalName: "J Microbiol Biotechnol"; date: "2017-02-28- 28"; 
            volume: "27"; issue: "2"; pages: "357-364"
COMMENT     Sequence Label: YSBE 559 : pNAT_6xHA
FEATURES             Location/Qualifiers
     source          1..5905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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        join(143..148,149..166,167..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(2180..2269)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             complement(2279..2368)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(2488..2505)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2488..2504)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2497..2519)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2642..2944
                     /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     2823..2842
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3293..4087
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3293..4087
                     /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(3347..3366)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3957..3976
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4105..4173,4174..4965)
                     /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(4323..4342)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5136..5724
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5625..5644
                     /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 caccatctat catcccaact tatcccgact cttcctcgtc
     2041 ccaccgccca ccacacacca cattactttc aggctcctga atacaacaca tcattcataa
     2101 gttcaagtat acgccaacat ctcttctttc gttataaata aaaagaaaga aagaaagaag
     2161 aacgattctt tctagcttaa gcgtaatctg gaacgtcata tggataggat cctgcatagt
     2221 ccgggacgtc atagggatag cccgcatagt caggaacatc gtatgggtat tctagacaag
     2281 cgtaatctgg aacgtcatat ggataggatc ctgcatagtc cgggacgtca tagggatagc
     2341 ccgcatagtc aggaacatcg tatgggtatg gcgcgccgca tgctgcggcc gcaagggcga
     2401 attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag ggcccaattc
     2461 gccctatagt gaatcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga
     2521 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     2581 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tatacgtacg
     2641 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
     2701 gagtgatatt attgacacgc cggggcgacg gatggtgatc cccctggcca gtgcacgtct
     2761 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
     2821 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
     2881 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     2941 ataaatgtca ggcatgagat tatcaaaaag gatcttcacc tagatccttt tcacgtagaa
     3001 agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg ctatctggac
     3061 aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta catggcgata
     3121 gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg gggcgccctc
     3181 tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg
     3241 atggcgcagg ggatcaagct ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3301 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3361 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3421 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     3481 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3541 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3601 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3661 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3721 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3781 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
     3841 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3901 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3961 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4021 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4081 cttctgaatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4141 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4201 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4261 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4321 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4381 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4441 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4501 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4561 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4621 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     4681 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4741 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4801 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     4861 ccctcccgta tcgtagttat ctacacgacg gggagccagg caactatgga tgaacgaaat
     4921 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     4981 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5041 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5101 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5161 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5221 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5281 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5341 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5401 accgggttgg attcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5461 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5521 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5581 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5641 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5701 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5761 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5821 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     5881 gaatcagtga gcgaggaagc ggaag
//
