LOCUS       sequence-443471-        9128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoterless (SwaI Cutsite) mRuby3:amdS fusion reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9128)
  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 9128)
  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: pLSPR24
FEATURES             Location/Qualifiers
     source          1..9128
                     /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"
     primer_bind     complement(6939..6959)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6939..6957)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6978..6994)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6978..6994)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6991..7013)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7002..7018
                     /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(7026..7032,7033..7050,7051..7056))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7071..7092
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7209..7226)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7380..7968)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7460..7479)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8139..8930,8931..8999))
                     /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     8762..8781
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9000..9104)
                     /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 ggtctctaag ggcgaggagc tcatcaagga
     3841 gaacatgcgt atgaaggtcg tcatggaggg ttctgtcaac ggccaccagt tcaagtgcac
     3901 tggtgagggt gagggcaggc cctacgaggg tgtccagacc atgaggatca aggtcatcga
     3961 gggtggtccc ctccctttcg ccttcgacat tcttgccact tctttcatgt acggctcccg
     4021 taccttcatc aagtaccccg ccgacatccc tgacttcttc aagcagtcct tccctgaggg
     4081 tttcacttgg gagagggtta ccaggtacga ggacggtggt gtcgtcaccg tcacccagga
     4141 cacctccctt gaggacggcg agctcgtcta caacgtcaag gtcaggggcg ttaacttccc
     4201 ctccaacggt cccgtcatgc agaagaagac caagggttgg gagcctaaca ctgagatgat
     4261 gtaccctgct gacggtggtc tcaggggtta cactgacatc gctctcaagg ttgacggtgg
     4321 tggccacctc cactgcaact tcgtcactac ttacaggtct aagaagaccg tcggcaacat
     4381 caagatgccc ggtgtccacg ccgttgacca ccgtctcgag aggatcgagg agtccgacaa
     4441 cgagacctac gttgtccaga gggaggtcgc tgttgccaag tactccaacc ttggtggtgg
     4501 catggacgag ctctacaagg gttctggcgg tggtggctcc ggcatgcctc agtcctggga
     4561 ggagctcgcc gctgacaagc gtgcccgtct cgctaagacc atccctgacg agtggaaggt
     4621 ccagaccctc cctgccgagg actccgttat tgacttccct aagaagtctg gcatcctttc
     4681 tgaggccgag ctcaagatca ctgaggcctc cgctgctgac cttgtctcca agctcgccgc
     4741 cggtgagttg acctctgtcg aggttaccct tgctttctgt aagcgagctg ctatcgccca
     4801 gcagttgact aactgcgccc acgagttctt ccctgacgcc gctctcgccc aggctaggga
     4861 gctcgacgag tactacgcta agcacaagag gcccgttggt cctctccacg gcctccccat
     4921 ctctctcaag gaccagcttc gagtcaaggg ctacgagact tctatgggct acatctcttg
     4981 gcttaacaag tacgacgagg gcgactctgt tctcactacc atgctccgta aggccggtgc
     5041 cgtcttctac gtcaagacct ctgtccccca gaccctcatg gtctgcgaga ctgtcaacaa
     5101 catcatcggc cgtaccgtca accctcgtaa caagaactgg tcttgcggcg gctcctctgg
     5161 tggtgagggt gccatcgttg gcattcgtgg tggcgtcatc ggtgttggta ccgacattgg
     5221 tggctctatt cgagtccccg ccgccttcaa cttcctctac ggtcttaggc cctcccacgg
     5281 ccgactcccc tacgctaaga tggccaactc catggagggt caggagaccg tccactccgt
     5341 tgtcggcccc attacccact ctgttgagga cctccgtctc ttcaccaagt ccgtcctcgg
     5401 tcaggagcct tggaagtacg actccaaggt catccccatg ccctggcgtc agtccgagtc
     5461 tgacattatt gcctccaaga tcaagaacgg cggcctcaac atcggctact acaacttcga
     5521 cggcaacgtc cttcctcacc ctcctatcct ccgtggcgtc gagaccaccg tcgccgctct
     5581 cgccaaggcc ggtcacaccg tcaccccctg gaccccttac aagcacgact tcggccacga
     5641 cctcatctcc cacatctacg ccgctgacgg ctccgccgac gttatgcgtg acatctccgc
     5701 ttccggcgag cccgccattc ctaacatcaa ggaccttctc aaccccaaca tcaaggctgt
     5761 taacatgaac gagctctggg acacccacct ccagaagtgg aactaccaga tggagtacct
     5821 tgagaagtgg cgagaggctg aggagaaggc cggcaaggag gtgagtagat cacaatttgt
     5881 aacaggttgg atgataagtc cccggtctaa catgtagcga cgacactgaa gctcacttct
     5941 tgtagctcga cgccatcatc gcccccatta cccctaccgc tgccgttcga cacgaccagt
     6001 tccgatacta cggctacgcc tctgtcatca acctcctcga cttcacctcc gtcgttgttc
     6061 ccgttacctt cgccgacaag aacatcgaca agaagaacga gtccttcaag gccgtttccg
     6121 agcttgacgc cctcgtccag gaggagtacg accccgaggc ctaccacggc gctcccgttg
     6181 ctgtccaggt tatcggtcga aggctctccg aggagaggac cttggccatt gctgaggagg
     6241 tcggcaagtt gctcggtaac gtcgtcactc cttagaaggt tgagcgcgta atagtgggtt
     6301 ggtcttgtct ctatccattc atctgtgaga catactgatt tcaacttttt catcagataa
     6361 taaggatggt gtgagataag caaggctggg ttatgaggtt tgggactaga tggtgtttcg
     6421 cgtctaaagg tgagtcttcg ttggttcaat cacattgatc tgatcgctga tcaattgtca
     6481 gcctctgaat gtgctcttga agaaaatggt tgtctgtaag aatccagaac caaaaacggt
     6541 catttcaagt atgtatcctc cgtgaagttc ctttagctct tgctaatatt ggactttagc
     6601 gtgctttagc caaagacagg gtcgcgaggt taagaggatg ggccaggaaa aatctaagtt
     6661 agatttgtca cacaaagaaa aagattgtct cagttttggt tttgtttggt ttgatttatg
     6721 cataggaaat atttatgatc gaaatcactc atttcctgct tcgtaaatat catctactat
     6781 atgcttcgta aatgtccatc tcctacatat tgtggatatg ataaatgcaa tcctctcgtc
     6841 tacactctcg gcttatgcct ttgtcttctt tgcgctcctt ttagccttct ttttattcct
     6901 agtcttcaag cccatcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg
     6961 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     7021 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     7081 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     7141 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     7201 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     7261 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     7321 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     7381 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     7441 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     7501 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     7561 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     7621 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     7681 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     7741 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     7801 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     7861 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     7921 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     7981 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     8041 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     8101 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     8161 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     8221 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     8281 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     8341 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     8401 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     8461 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     8521 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     8581 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     8641 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     8701 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     8761 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     8821 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     8881 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     8941 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     9001 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     9061 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     9121 gtgccacc
//
