LOCUS       sequence-176997-        6349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for tagging proteins in Cryptococcus neoformans; 6xHA tag, 
            HYG selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6349)
  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 6349)
  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 610 : pHYG_6xHA
FEATURES             Location/Qualifiers
     source          1..6349
                     /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             1019..2044
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1019..2044
                     /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(2624..2713)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             complement(2723..2812)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(2932..2949)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2932..2948)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2941..2963)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3086..3388
                     /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     3267..3286
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3737..4531
                     /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             3737..4531
                     /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(3791..3810)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4401..4420
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4549..4617,4618..5409)
                     /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(4767..4786)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5580..6168
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6069..6088
                     /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 gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     1081 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     1141 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     1201 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
     1261 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     1321 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
     1381 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     1441 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     1501 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     1561 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     1621 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     1681 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     1741 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     1801 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     1861 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     1921 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     1981 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     2041 ataggtgaag gcggtaaggg gttaattttc cttagagggt gtatatatac atattagaga
     2101 agtgatacaa ttttagcaca ctgcgaattc gagacagaca tcgtgtcaat catctttttt
     2161 gacattatat gcccattaat ctatctacag acaacaatac catccttccc accctcagca
     2221 acgccgttga atcctcagga tcttcatggc tccaagcttg gtaccgagct cggatccact
     2281 agtaacggcc gccagtgtgc tggaattcgc ccttgtacca atctatccct ctctccgaac
     2341 aatctccaca ttccctccct tctgctccct gaacccgcaa cacgagccgt ccacacttct
     2401 tctcaacttt gcgcttctca acttttgctt atgcaatctt gctccaccat ctatcatccc
     2461 aacttatccc gactcttcct cgtcccaccg cccaccacac accacattac tttcaggctc
     2521 ctgaatacaa cacatcattc ataagttcaa gtatacgcca acatctcttc tttcgttata
     2581 aataaaaaga aagaaagaaa gaagaacgat tctttctagc ttaagcgtaa tctggaacgt
     2641 catatggata ggatcctgca tagtccggga cgtcataggg atagcccgca tagtcaggaa
     2701 catcgtatgg gtattctaga caagcgtaat ctggaacgtc atatggatag gatcctgcat
     2761 agtccgggac gtcataggga tagcccgcat agtcaggaac atcgtatggg tatggcgcgc
     2821 cgcatgctgc ggccgcaagg gcgaattctg cagatatcca tcacactggc ggccgctcga
     2881 gcatgcatct agagggccca attcgcccta tagtgaatcg tattacaatt cactggccgt
     2941 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     3001 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     3061 acagttgcgc agcctatacg tacggcagtt taaggtttac acctataaaa gagagagccg
     3121 ttatcgtctg tttgtggatg tacagagtga tattattgac acgccggggc gacggatggt
     3181 gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt
     3241 ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt
     3301 ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc
     3361 cattaacctg atgttctggg gaatataaat gtcaggcatg agattatcaa aaaggatctt
     3421 cacctagatc cttttcacgt agaaagccag tccgcagaaa cggtgctgac cccggatgaa
     3481 tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc
     3541 ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag caagcgaacc
     3601 ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat
     3661 ggctttcttg ccgccaagga tctgatggcg caggggatca agctctgatc aagagacagg
     3721 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     3781 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     3841 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3901 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3961 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4021 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4081 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4141 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4201 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4261 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4321 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4381 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4441 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4501 cttctatcgc cttcttgacg agttcttctg aattgaaaaa ggaagagtat gagtattcaa
     4561 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4621 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4681 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4741 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4801 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4861 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     4921 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     4981 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5041 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5101 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5161 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5221 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5281 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagc
     5341 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5401 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5461 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5521 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5581 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5641 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5701 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5761 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5821 gccagtggcg ataagtcgtg tcttaccggg ttggattcaa gacgatagtt accggataag
     5881 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5941 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6001 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6061 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6121 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6181 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6241 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6301 cgcagccgaa cgaccgagcg cagcgaatca gtgagcgagg aagcggaag
//
