LOCUS       sequence-176999-        6637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for tagging proteins in Cryptococcus neoformans; GFP tag, 
            Neo selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6637)
  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 6637)
  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 508 : pNEO_GFP
FEATURES             Location/Qualifiers
     source          1..6637
                     /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..1831
                     /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="MSMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(join(2384..3097,3098..3100))
                     /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(2429..2450)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2778..2797
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3037..3058
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3220..3237)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3220..3236)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3229..3251)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3374..3676
                     /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     3555..3574
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4025..4819
                     /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             4025..4819
                     /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             join(4837..4905,4906..5697)
                     /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(5055..5074)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5868..6456
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6357..6376
                     /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 gtaccgagct cggatccgca tgcaggattc gagtggcatg gtgtgcactg agtgtatggt
      301 tgtcggagga gaggatgatg gtaacaacaa tagcagcaac gtcactcgac gcgcgtccgg
      361 tgtgccacac ggggtaacgc cgagtcgccg tcagggtcgc cgagaccact ctcacagcgt
      421 caccgttggc accagctcag cttacagctt ctatcctccg ccagcatcca catacatccc
      481 ctataccgca tcccccaccc actgcccaag gtgagtcatc ttcccgcccc cttcccttgc
      541 ccgccactca gtcctccatc ctccactaat ccaccttatc gcacccaccg cctatcgcac
      601 atccgagcac aatgctgggc ctgccagggg ctgctagatg gtgctctccc cacgctgatc
      661 tgcatgccgg ccattggatc atgggtgcta ggtgctgggt gctggatgtt ggatgctgga
      721 tgctgggtgc acgcttggtc atttccttcc aggattgacg gtcgccgaga ggacgacgtg
      781 gcgttcgaca acgagggccg atagcaccgc atcgcctcga cctgcatcca tctcgccttg
      841 tccttttggt gcaacaatcc atccgtgctg gtgccacacg catagctgga agagatggat
      901 gtgcgttgaa cagagctgcc gtcaggactt tttggtgcac ggaccctatt gtcctcccca
      961 atcttcaccg cgtctcctaa tatgcagcct ctttgctaat tgtctttttc cattagtaaa
     1021 ctcgcccaac atgtctatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     1081 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     1141 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     1201 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     1261 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     1321 cgaagtgccg gggcaggatc tcctgtcatc ccaccttgct cctgccgaga aagtatccat
     1381 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     1441 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     1501 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     1561 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     1621 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     1681 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     1741 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     1801 cttctatcgc cttcttgacg agttcttctg agtgaaggcg gtaaggggtt aattttcctt
     1861 agagggtgta tatatacata ttagagaagt gatacaattt tagcacactg cgaattcgag
     1921 acagacatcg tgtcaatcat cttttttgac attatatgcc cattaatcta tctacagaca
     1981 acaataccat ccttcccacc ctcagcaacg ccgttgaatc ctcaggatct tcatggctcc
     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 cttttacttg tacagctcgt
     2401 ccatgccgtg agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct
     2461 tctcgttggg gtctttgctc agggcggact gggtgctcag gtagtggttg tcgggcagca
     2521 gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc
     2581 cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg
     2641 ccatgatata gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc
     2701 cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga
     2761 acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct
     2821 ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt
     2881 agcggctgaa gcactgcacg ccgtaggtga aggtggtcac gagggtgggc cagggcacgg
     2941 gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct
     3001 cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga
     3061 tgggcaccac cccggtgaac agctcctcgc ccttgctcac agagccaccg ccacctgcgg
     3121 ccgcaagggc gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag
     3181 agggcccaat tcgccctata gtgaatcgta ttacaattca ctggccgtcg ttttacaacg
     3241 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     3301 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     3361 cctatacgta cggcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt
     3421 tgtggatgta cagagtgata ttattgacac gccggggcga cggatggtga tccccctggc
     3481 cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg
     3541 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg
     3601 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat
     3661 gttctgggga atataaatgt caggcatgag attatcaaaa aggatcttca cctagatcct
     3721 tttcacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg
     3781 ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct
     3841 tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc
     3901 tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc
     3961 gccaaggatc tgatggcgca ggggatcaag ctctgatcaa gagacaggat gaggatcgtt
     4021 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4081 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4141 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4201 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4261 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4321 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4381 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4441 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4501 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     4561 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4621 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4681 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4741 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4801 tcttgacgag ttcttctgaa ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4861 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4921 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4981 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5041 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5101 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5161 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5221 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5281 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5341 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5401 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5461 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5521 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5581 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagcca ggcaactatg
     5641 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5701 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5761 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5821 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5881 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5941 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6001 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6061 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6121 aagtcgtgtc ttaccgggtt ggattcaaga cgatagttac cggataaggc gcagcggtcg
     6181 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6241 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6301 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6361 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6421 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6481 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6541 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6601 accgagcgca gcgaatcagt gagcgaggaa gcggaag
//
