LOCUS       sequence-443463-        9125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoterless (SwaI Cutsite) mNeonGreen:amdS fusion reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9125)
  AUTHORS   Phillips-Rose LS, Yu CK, West NP, Fraser JA
  TITLE     A Chimeric ORF Fusion Phenotypic Reporter for Cryptococcus 
            neoformans.
  JOURNAL   J Fungi (Basel). 2024 Aug 12;10(8):567. doi: 10.3390/jof10080567.
  PUBMED    39194893
REFERENCE   2  (bases 1 to 9125)
  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"; journalName: "J Fungi 
            (Basel)."; date: "2024-08-12"; volume: "10(8):567. doi"; pages: " 
            10.3390/jof10080567"
COMMENT     Sequence Label: pLSPR25
FEATURES             Location/Qualifiers
     source          1..9125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1823..2623)
                     /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"
     primer_bind     complement(1934..1953)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2544..2563
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3713..3735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3727..3744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3728..3744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3754..3773
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3754..3772
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4064..4073
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6936..6956)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6936..6954)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6975..6991)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6975..6991)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6988..7010)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6999..7015
                     /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)."
     promoter        complement(join(7023..7029,7030..7047,7048..7053))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7068..7089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7206..7223)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7377..7965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7457..7476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8136..8927,8928..8996))
                     /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     8759..8778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8997..9101)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgacgcgccc tgtagcggca agatctgccc atagacttca aatcatggaa ttggacgttt
       61 atcttcttgc tttggatatc caccctatcg tacaatcttg gcactttgga tcatacgagg
      121 cactttgcgg actagggaat ggttatcatc aatttcttgg acctctccat gacacctggc
      181 atcgtcgagc taccacagcc ccctgaattc ttaaaggcga accccccttc ctaggaacct
      241 tgagcgctgg actggatggc actctgagac cgcctatgct taaggacgcg agcttaccct
      301 tccattgatg gaagtcgatc atttatgata atagaacacg gcggaacttg actttacagg
      361 ccaagggaag caacaggaga agaagctaag gttataacag gctcagcata tttgtatata
      421 cggtgccatt caacgctgaa cactttgtac tcacggtctg cccgttgaat aacaccatct
      481 gctgatgatc atcccaattt gtgagtctta tttgccattg atcctggtgt ataaccgata
      541 ctgaagccct ttaggtctga tcagttatag acgtggtata ttcattcttc ctagattagc
      601 tatgccgaaa gagatggata ccttgatgtg actggaatgc catgaagcag gagattgtag
      661 cagctcaaat tggagaagga tgttgcaacc gtcggccgtg aaggtcttgt catatctttg
      721 tctgcctggg agaatatgta caagtgcccc gatgttactg atggaaaaca tcgtgtggcg
      781 cgccacgata cttgtgttaa ttaaaccaaa ccagtctgtg ccaagtggat tgcttatgca
      841 tcgagtggcg agcccactga cctctgggcc ccttttttga actataaaaa gcggccaagt
      901 ctgatttcgg tctcacaatc atcaacatcg acttctcttt tcgcctcgat caacctaaac
      961 aacggtcact cagccatgaa ctggacaatg gaatatttcg ccttgccttg gatctttggc
     1021 ctctagactc aatcaacgtc aggataaagg tgagcatagg ttgctcactc actgtagcct
     1081 ttttttccct tcttcttggt ccagatggtt cccaaggctc tataaagtgt atctgtcagt
     1141 tccacttcct tactccgaag cgattcagag ttgtaaagtg cgtctgaccc cctgattata
     1201 tctcaaagtc ccttcacaaa ttcagacatg atgcgataaa gtgccggatt gtcattcgcg
     1261 tcttgtgact gtatagaaag tgccggcatc ctcttttctt ttcctctttg gccgagcgac
     1321 tcagagttgt gaagcctgct ggtatctgtc tgttcccttg ccctgataaa gaatatcgaa
     1381 gagcatttct ttgattcaca atacgttgac gaatggtgtg atggatccta agcagcgcca
     1441 gccctttgaa cttgtgtcga aaaaaaaaaa aaaacagcat atgatacgta aagggaagaa
     1501 aaggatggag ctgactgttt tgacaggcat aattttgagc gtgcttcatt ggccggttta
     1561 tctgtattaa cacggaagag atgtagaaac tagcttcctg gtttcagaga caaggagcca
     1621 tgaagatcct gaggattcaa cggcgttgct gagggtggga aggatggtat tgttgtctgt
     1681 agatagatta atgggcatat aatgtcaaaa aagatgattg acacgatgtc tgtctcgaat
     1741 tcgcagtgtg ctaaaattgt atcacttctc taatatgtat atatacaccc tctaaggaaa
     1801 attaacccct taccgccttc actcagaaga actcgtcaag aaggcgatag aaggcgatgc
     1861 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc
     1921 caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac
     1981 ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca
     2041 agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgcgc gccttgagcc
     2101 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
     2161 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
     2221 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
     2281 ctttctcggc aggagcaagg tgggatgaca ggagatcctg ccccggcact tcgcccaata
     2341 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg
     2401 tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca
     2461 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
     2521 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg
     2581 ccggagaacc tgcgtgcaat ccatcttgtt caatcataga catgttgggc gagtttacta
     2641 atggaaaaag acaattagca aagaggctgc atattaggag acgcggtgaa gattggggag
     2701 gacaataggg tccgtgcacc aaaaagtcct gacggcagct ctgttcaacg cacatccatc
     2761 tcttccagct atgcgtgtgg caccagcacg gatggattgt tgcaccaaaa ggacaaggcg
     2821 agatggatgc aggtcgaggc gatgcggtgc tatcggccct cgttgtcgaa cgccacgtcg
     2881 tcctctcggc gaccgtcaat cctggaagga aatgaccaag cgtgcaccca gcatccagca
     2941 tccaacatcc agcacccagc acctagcacc catgatccaa tggccggcat gcagatcagc
     3001 gtggggagag caccatctag cagcccctgg caggcccagc attgtgctcg gatgtgcgat
     3061 aggcggtggg tgcgataagg tggattagtg gaggatggag gactgagtgg cgggcaaggg
     3121 aagggggcgg gaagatgact caccttgggc agtgggtggg ggatgcggta taggggatgt
     3181 atgtggatgc tggcggagga tagaagctgt aagctgagct ggtgccaacg gtgacgctgt
     3241 gagagtggtc tcggcgaccc tgacggcgac tcggcgttac cccgtgtggc acaccggacg
     3301 cgcgtcgagt gacgttgctg ctattgttgt taccatcatc ctctcctccg acaaccatac
     3361 actcagtgca caccatgcca ctcgaatcct gcatgcttat gtgagtcctc cccttcccgt
     3421 gcgccgctct ccagctcaca tcctcgcagc catggactca accctatctc ggtctattct
     3481 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaaatg agctgattta
     3541 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc
     3601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     3661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     3721 cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg
     3781 ggtaccgggc cccccctcga atttaaatat ggtctccaag ggcgaggagg acaacatggc
     3841 ctctctccct gccactcacg agttgcacat cttcggctcc atcaacggtg tcgacttcga
     3901 catggtcggt cagggcaccg gcaaccctaa cgacggttac gaggagttga accttaagtc
     3961 caccaagggt gacctccagt tctccccctg gattcttgtc cctcacatcg gctacggctt
     4021 ccaccagtac cttccctacc ctgacggcat gtcccctttc caggccgcca tggttgacgg
     4081 ctccggttac caggtccacc gtactatgca gttcgaggac ggtgcctccc ttactgttaa
     4141 ctaccgttac acctacgagg gttcccacat caagggtgag gcccaggtca agggcactgg
     4201 tttccctgct gacggtcctg tcatgaccaa ctcccttacc gctgccgact ggtgcaggtc
     4261 caagaagact taccccaacg acaagaccat catctccacc ttcaagtggt cctacaccac
     4321 tggtaacggc aagcgttacc gatccactgc ccgaaccacc tacaccttcg ccaagcctat
     4381 ggccgctaac taccttaaga accagcccat gtacgttttc cgtaagaccg agctcaagca
     4441 ctccaagacc gagctcaact tcaaggagtg gcagaaggcc ttcaccgacg tcatgggcat
     4501 ggacgagctt tacaagggtt ctggcggtgg tggctccggc atgcctcagt cctgggagga
     4561 gctcgccgct gacaagcgtg cccgtctcgc taagaccatc cctgacgagt ggaaggtcca
     4621 gaccctccct gccgaggact ccgttattga cttccctaag aagtctggca tcctttctga
     4681 ggccgagctc aagatcactg aggcctccgc tgctgacctt gtctccaagc tcgccgccgg
     4741 tgagttgacc tctgtcgagg ttacccttgc tttctgtaag cgagctgcta tcgcccagca
     4801 gttgactaac tgcgcccacg agttcttccc tgacgccgct ctcgcccagg ctagggagct
     4861 cgacgagtac tacgctaagc acaagaggcc cgttggtcct ctccacggcc tccccatctc
     4921 tctcaaggac cagcttcgag tcaagggcta cgagacttct atgggctaca tctcttggct
     4981 taacaagtac gacgagggcg actctgttct cactaccatg ctccgtaagg ccggtgccgt
     5041 cttctacgtc aagacctctg tcccccagac cctcatggtc tgcgagactg tcaacaacat
     5101 catcggccgt accgtcaacc ctcgtaacaa gaactggtct tgcggcggct cctctggtgg
     5161 tgagggtgcc atcgttggca ttcgtggtgg cgtcatcggt gttggtaccg acattggtgg
     5221 ctctattcga gtccccgccg ccttcaactt cctctacggt cttaggccct cccacggccg
     5281 actcccctac gctaagatgg ccaactccat ggagggtcag gagaccgtcc actccgttgt
     5341 cggccccatt acccactctg ttgaggacct ccgtctcttc accaagtccg tcctcggtca
     5401 ggagccttgg aagtacgact ccaaggtcat ccccatgccc tggcgtcagt ccgagtctga
     5461 cattattgcc tccaagatca agaacggcgg cctcaacatc ggctactaca acttcgacgg
     5521 caacgtcctt cctcaccctc ctatcctccg tggcgtcgag accaccgtcg ccgctctcgc
     5581 caaggccggt cacaccgtca ccccctggac cccttacaag cacgacttcg gccacgacct
     5641 catctcccac atctacgccg ctgacggctc cgccgacgtt atgcgtgaca tctccgcttc
     5701 cggcgagccc gccattccta acatcaagga ccttctcaac cccaacatca aggctgttaa
     5761 catgaacgag ctctgggaca cccacctcca gaagtggaac taccagatgg agtaccttga
     5821 gaagtggcga gaggctgagg agaaggccgg caaggaggtg agtagatcac aatttgtaac
     5881 aggttggatg ataagtcccc ggtctaacat gtagcgacga cactgaagct cacttcttgt
     5941 agctcgacgc catcatcgcc cccattaccc ctaccgctgc cgttcgacac gaccagttcc
     6001 gatactacgg ctacgcctct gtcatcaacc tcctcgactt cacctccgtc gttgttcccg
     6061 ttaccttcgc cgacaagaac atcgacaaga agaacgagtc cttcaaggcc gtttccgagc
     6121 ttgacgccct cgtccaggag gagtacgacc ccgaggccta ccacggcgct cccgttgctg
     6181 tccaggttat cggtcgaagg ctctccgagg agaggacctt ggccattgct gaggaggtcg
     6241 gcaagttgct cggtaacgtc gtcactcctt agaaggttga gcgcgtaata gtgggttggt
     6301 cttgtctcta tccattcatc tgtgagacat actgatttca actttttcat cagataataa
     6361 ggatggtgtg agataagcaa ggctgggtta tgaggtttgg gactagatgg tgtttcgcgt
     6421 ctaaaggtga gtcttcgttg gttcaatcac attgatctga tcgctgatca attgtcagcc
     6481 tctgaatgtg ctcttgaaga aaatggttgt ctgtaagaat ccagaaccaa aaacggtcat
     6541 ttcaagtatg tatcctccgt gaagttcctt tagctcttgc taatattgga ctttagcgtg
     6601 ctttagccaa agacagggtc gcgaggttaa gaggatgggc caggaaaaat ctaagttaga
     6661 tttgtcacac aaagaaaaag attgtctcag ttttggtttt gtttggtttg atttatgcat
     6721 aggaaatatt tatgatcgaa atcactcatt tcctgcttcg taaatatcat ctactatatg
     6781 cttcgtaaat gtccatctcc tacatattgt ggatatgata aatgcaatcc tctcgtctac
     6841 actctcggct tatgcctttg tcttctttgc gctcctttta gccttctttt tattcctagt
     6901 cttcaagccc atcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc
     6961 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     7021 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     7081 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     7141 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     7201 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     7261 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     7321 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     7381 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     7441 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     7501 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     7561 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     7621 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     7681 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     7741 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     7801 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     7861 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     7921 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     7981 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     8041 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     8101 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     8161 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     8221 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     8281 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     8341 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     8401 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     8461 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     8521 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     8581 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     8641 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     8701 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     8761 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     8821 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     8881 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     8941 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     9001 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     9061 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     9121 ccacc
//
