LOCUS       sequence-177008-        6415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for tagging proteins in Cryptococcus neoformans; mCherry 
            tag, NAt selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6415)
  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 6415)
  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 596 : pNAT_mCherry
FEATURES             Location/Qualifiers
     source          1..6415
                     /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(2172..2879)
                     /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(2450..2469)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2719..2737
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2998..3015)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2998..3014)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3007..3029)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3152..3454
                     /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     3333..3352
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3803..4597
                     /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             3803..4597
                     /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(3857..3876)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4467..4486
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4615..4683,4684..5475)
                     /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(4833..4852)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5646..6234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6135..6154
                     /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 agtaccaatc tatccctctc tccgaacaat ctccacattc cctcccttct
     1921 gctccctgaa cccgcaacac gagccgtcca cacttcttct caactttgcg cttctcaact
     1981 tttgcttatg caatcttgct ccaccatcta tcatcccaac ttatcccgac tcttcctcgt
     2041 cccaccgccc accacacacc acattacttt caggctcctg aatacaacac atcattcata
     2101 agttcaagta tacgccaaca tctcttcttt cgttataaat aaaaagaaag aaagaaagaa
     2161 gaacgattct tttacttgta cagctcgtcc atgccgccgg tggagtggcg gccctcggcg
     2221 cgttcgtact gttccacgat ggtgtagtcc tcgttgtggg aggtgatgtc caacttgatg
     2281 ttgacgttgt aggcgccggg cagctgcacg ggcttcttgg ccttgtaggt ggtcttgacc
     2341 tcagcgtcgt agtggccgcc gtccttcagc ttcagcctct gcttgatctc gcccttcagg
     2401 gcgccgtcct cggggtacat ccgctcggag gaggcctccc agcccatggt cttcttctgc
     2461 attacggggc cgtcggaggg gaagttggtg ccgcgcagct tcaccttgta gatgaactcg
     2521 ccgtcctgca gggaggagtc ctgggtcacg gtcaccacgc cgccgtcctc gaagttcatc
     2581 acgcgctccc acttgaagcc ctcggggaag gacagcttca agtagtcggg gatgtcggcg
     2641 gggtgcttca cgtaggcctt ggagccgtac atgaactgag gggacaggat gtcccaggcg
     2701 aagggcaggg ggccaccctt ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg
     2761 cggccctcgc cctcgccctc gatctcgaac tcgtggccgt tcacggagcc ctccatgtgc
     2821 accttgaagc gcatgaactc cttgatgatg gccatgttat cctcctcgcc cttgctcaca
     2881 gagccaccgc cacctgcggc cgcagggcga attctgcaga tatccatcac actggcggcc
     2941 gctcgagcat gcatctagag ggcccaattc gccctatagt gaatcgtatt acaattcact
     3001 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     3061 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     3121 ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga
     3181 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg
     3241 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta
     3301 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt
     3361 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
     3421 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag
     3481 gatcttcacc tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg
     3541 gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca
     3601 ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag
     3661 cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa
     3721 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga
     3781 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3841 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3901 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3961 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     4021 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4081 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4141 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4201 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4261 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4321 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4381 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4441 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4501 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4561 catcgccttc tatcgccttc ttgacgagtt cttctgaatt gaaaaaggaa gagtatgagt
     4621 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4681 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4741 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4801 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4861 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4921 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4981 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5041 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5101 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5161 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5221 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5281 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5341 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5401 gggagccagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5461 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5521 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5581 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5641 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5701 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5761 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5821 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5881 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg attcaagacg atagttaccg
     5941 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6001 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6061 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6121 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6181 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6241 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6301 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     6361 gctcgccgca gccgaacgac cgagcgcagc gaatcagtga gcgaggaagc ggaag
//
