LOCUS       sequence-176994-        6868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for tagging proteins in Cryptococcus neoformans; GFP tag, 
            HYG selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6868)
  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 6868)
  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 608 : pHYG_GFP
FEATURES             Location/Qualifiers
     source          1..6868
                     /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(join(2615..3328,3329..3331))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2660..2681)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3009..3028
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3268..3289
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3451..3468)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3451..3467)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3460..3482)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3605..3907
                     /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     3786..3805
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4256..5050
                     /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             4256..5050
                     /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(4310..4329)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4920..4939
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(5068..5136,5137..5928)
                     /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(5286..5305)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6099..6687
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6588..6607
                     /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 tcttttactt gtacagctcg tccatgccgt
     2641 gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg
     2701 ggtctttgct cagggcggac tgggtgctca ggtagtggtt gtcgggcagc agcacggggc
     2761 cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga
     2821 tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat
     2881 agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct
     2941 tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct
     3001 cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc
     3061 cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcggctga
     3121 agcactgcac gccgtaggtg aaggtggtca cgagggtggg ccagggcacg ggcagcttgc
     3181 cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc
     3241 cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca
     3301 ccccggtgaa cagctcctcg cccttgctca cagagccacc gccacctgcg gccgcaaggg
     3361 cgaattctgc agatatccat cacactggcg gccgctcgag catgcatcta gagggcccaa
     3421 ttcgccctat agtgaatcgt attacaattc actggccgtc gttttacaac gtcgtgactg
     3481 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     3541 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctatacgt
     3601 acggcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt
     3661 acagagtgat attattgaca cgccggggcg acggatggtg atccccctgg ccagtgcacg
     3721 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag
     3781 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt
     3841 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg
     3901 aatataaatg tcaggcatga gattatcaaa aaggatcttc acctagatcc ttttcacgta
     3961 gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg
     4021 gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg
     4081 atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc
     4141 ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat
     4201 ctgatggcgc aggggatcaa gctctgatca agagacagga tgaggatcgt ttcgcatgat
     4261 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4321 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4381 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4441 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4501 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4561 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4621 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4681 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4741 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4801 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4861 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4921 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4981 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     5041 gttcttctga attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5101 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5161 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5221 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5281 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5341 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5401 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5461 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5521 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5581 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5641 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5701 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5761 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5821 aagccctccc gtatcgtagt tatctacacg acggggagcc aggcaactat ggatgaacga
     5881 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5941 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6001 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6061 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6121 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6181 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6241 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6301 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6361 cttaccgggt tggattcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6421 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6481 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6541 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6601 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6661 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6721 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6781 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6841 agcgaatcag tgagcgagga agcggaag
//
