LOCUS       sequence-443444-        9125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoterless (SwaI Cutsite) amdS:mNeonGreen 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: pLSPR23
FEATURES             Location/Qualifiers
     source          1..9125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1824..2624)
                     /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(1935..1954)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2545..2564
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3714..3736
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3728..3745
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3729..3745
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3755..3774
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3755..3773
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5818..5827
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6937..6957)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6937..6955)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6976..6992)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6976..6992)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6989..7011)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7000..7016
                     /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(7024..7030,7031..7048,7049..7054))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7069..7090
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7207..7224)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7378..7966)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7458..7477)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8137..8928,8929..8997))
                     /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     8760..8779
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8998..9102)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc aagatctgcc catagacttc aaatcatgga attggacgtt
       61 tatcttcttg ctttggatat ccaccctatc gtacaatctt ggcactttgg atcatacgag
      121 gcactttgcg gactagggaa tggttatcat caatttcttg gacctctcca tgacacctgg
      181 catcgtcgag ctaccacagc cccctgaatt cttaaaggcg aacccccctt cctaggaacc
      241 ttgagcgctg gactggatgg cactctgaga ccgcctatgc ttaaggacgc gagcttaccc
      301 ttccattgat ggaagtcgat catttatgat aatagaacac ggcggaactt gactttacag
      361 gccaagggaa gcaacaggag aagaagctaa ggttataaca ggctcagcat atttgtatat
      421 acggtgccat tcaacgctga acactttgta ctcacggtct gcccgttgaa taacaccatc
      481 tgctgatgat catcccaatt tgtgagtctt atttgccatt gatcctggtg tataaccgat
      541 actgaagccc tttaggtctg atcagttata gacgtggtat attcattctt cctagattag
      601 ctatgccgaa agagatggat accttgatgt gactggaatg ccatgaagca ggagattgta
      661 gcagctcaaa ttggagaagg atgttgcaac cgtcggccgt gaaggtcttg tcatatcttt
      721 gtctgcctgg gagaatatgt acaagtgccc cgatgttact gatggaaaac atcgtgtggc
      781 gcgccacgat acttgtgtta attaaaccaa accagtctgt gccaagtgga ttgcttatgc
      841 atcgagtggc gagcccactg acctctgggc cccttttttg aactataaaa agcggccaag
      901 tctgatttcg gtctcacaat catcaacatc gacttctctt ttcgcctcga tcaacctaaa
      961 caacggtcac tcagccatga actggacaat ggaatatttc gccttgcctt ggatctttgg
     1021 cctctagact caatcaacgt caggataaag gtgagcatag gttgctcact cactgtagcc
     1081 tttttttccc ttcttcttgg tccagatggt tcccaaggct ctataaagtg tatctgtcag
     1141 ttccacttcc ttactccgaa gcgattcaga gttgtaaagt gcgtctgacc ccctgattat
     1201 atctcaaagt cccttcacaa attcagacat gatgcgataa agtgccggat tgtcattcgc
     1261 gtcttgtgac tgtatagaaa gtgccggcat cctcttttct tttcctcttt ggccgagcga
     1321 ctcagagttg tgaagcctgc tggtatctgt ctgttccctt gccctgataa agaatatcga
     1381 agagcatttc tttgattcac aatacgttga cgaatggtgt gatggatcct aagcagcgcc
     1441 agccctttga acttgtgtcg aaaaaaaaaa aaaaacagca tatgatacgt aaagggaaga
     1501 aaaggatgga gctgactgtt ttgacaggca taattttgag cgtgcttcat tggccggttt
     1561 atctgtatta acacggaaga gatgtagaaa ctagcttcct ggtttcagag acaaggagcc
     1621 atgaagatcc tgaggattca acggcgttgc tgagggtggg aaggatggta ttgttgtctg
     1681 tagatagatt aatgggcata taatgtcaaa aaagatgatt gacacgatgt ctgtctcgaa
     1741 ttcgcagtgt gctaaaattg tatcacttct ctaatatgta tatatacacc ctctaaggaa
     1801 aattaacccc ttaccgcctt cactcagaag aactcgtcaa gaaggcgata gaaggcgatg
     1861 cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg
     1921 ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
     1981 cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc
     2041 aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc
     2101 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg
     2161 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
     2221 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
     2281 actttctcgg caggagcaag gtgggatgac aggagatcct gccccggcac ttcgcccaat
     2341 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc
     2401 gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac
     2461 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
     2521 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
     2581 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatag acatgttggg cgagtttact
     2641 aatggaaaaa gacaattagc aaagaggctg catattagga gacgcggtga agattgggga
     2701 ggacaatagg gtccgtgcac caaaaagtcc tgacggcagc tctgttcaac gcacatccat
     2761 ctcttccagc tatgcgtgtg gcaccagcac ggatggattg ttgcaccaaa aggacaaggc
     2821 gagatggatg caggtcgagg cgatgcggtg ctatcggccc tcgttgtcga acgccacgtc
     2881 gtcctctcgg cgaccgtcaa tcctggaagg aaatgaccaa gcgtgcaccc agcatccagc
     2941 atccaacatc cagcacccag cacctagcac ccatgatcca atggccggca tgcagatcag
     3001 cgtggggaga gcaccatcta gcagcccctg gcaggcccag cattgtgctc ggatgtgcga
     3061 taggcggtgg gtgcgataag gtggattagt ggaggatgga ggactgagtg gcgggcaagg
     3121 gaagggggcg ggaagatgac tcaccttggg cagtgggtgg gggatgcggt ataggggatg
     3181 tatgtggatg ctggcggagg atagaagctg taagctgagc tggtgccaac ggtgacgctg
     3241 tgagagtggt ctcggcgacc ctgacggcga ctcggcgtta ccccgtgtgg cacaccggac
     3301 gcgcgtcgag tgacgttgct gctattgttg ttaccatcat cctctcctcc gacaaccata
     3361 cactcagtgc acaccatgcc actcgaatcc tgcatgctta tgtgagtcct ccccttcccg
     3421 tgcgccgctc tccagctcac atcctcgcag ccatggactc aaccctatct cggtctattc
     3481 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaaat gagctgattt
     3541 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc attcgccatt
     3601 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     3661 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     3721 acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt
     3781 gggtaccggg ccccccctcg aatttaaata tgcctcagtc ctgggaggag ctcgccgctg
     3841 acaagcgtgc ccgtctcgct aagaccatcc ctgacgagtg gaaggtccag accctccctg
     3901 ccgaggactc cgttattgac ttccctaaga agtctggcat cctttctgag gccgagctca
     3961 agatcactga ggcctccgct gctgaccttg tctccaagct cgccgccggt gagttgacct
     4021 ctgtcgaggt tacccttgct ttctgtaagc gagctgctat cgcccagcag ttgactaact
     4081 gcgcccacga gttcttccct gacgccgctc tcgcccaggc tagggagctc gacgagtact
     4141 acgctaagca caagaggccc gttggtcctc tccacggcct ccccatctct ctcaaggacc
     4201 agcttcgagt caagggctac gagacttcta tgggctacat ctcttggctt aacaagtacg
     4261 acgagggcga ctctgttctc actaccatgc tccgtaaggc cggtgccgtc ttctacgtca
     4321 agacctctgt cccccagacc ctcatggtct gcgagactgt caacaacatc atcggccgta
     4381 ccgtcaaccc tcgtaacaag aactggtctt gcggcggctc ctctggtggt gagggtgcca
     4441 tcgttggcat tcgtggtggc gtcatcggtg ttggtaccga cattggtggc tctattcgag
     4501 tccccgccgc cttcaacttc ctctacggtc ttaggccctc ccacggccga ctcccctacg
     4561 ctaagatggc caactccatg gagggtcagg agaccgtcca ctccgttgtc ggccccatta
     4621 cccactctgt tgaggacctc cgtctcttca ccaagtccgt cctcggtcag gagccttgga
     4681 agtacgactc caaggtcatc cccatgccct ggcgtcagtc cgagtctgac attattgcct
     4741 ccaagatcaa gaacggcggc ctcaacatcg gctactacaa cttcgacggc aacgtccttc
     4801 ctcaccctcc tatcctccgt ggcgtcgaga ccaccgtcgc cgctctcgcc aaggccggtc
     4861 acaccgtcac cccctggacc ccttacaagc acgacttcgg ccacgacctc atctcccaca
     4921 tctacgccgc tgacggctcc gccgacgtta tgcgtgacat ctccgcttcc ggcgagcccg
     4981 ccattcctaa catcaaggac cttctcaacc ccaacatcaa ggctgttaac atgaacgagc
     5041 tctgggacac ccacctccag aagtggaact accagatgga gtaccttgag aagtggcgag
     5101 aggctgagga gaaggccggc aaggaggtga gtagatcaca atttgtaaca ggttggatga
     5161 taagtccccg gtctaacatg tagcgacgac actgaagctc acttcttgta gctcgacgcc
     5221 atcatcgccc ccattacccc taccgctgcc gttcgacacg accagttccg atactacggc
     5281 tacgcctctg tcatcaacct cctcgacttc acctccgtcg ttgttcccgt taccttcgcc
     5341 gacaagaaca tcgacaagaa gaacgagtcc ttcaaggccg tttccgagct tgacgccctc
     5401 gtccaggagg agtacgaccc cgaggcctac cacggcgctc ccgttgctgt ccaggttatc
     5461 ggtcgaaggc tctccgagga gaggaccttg gccattgctg aggaggtcgg caagttgctc
     5521 ggtaacgtcg tcactcctgg ttctggcggt ggtggctccg gcatggtctc caagggcgag
     5581 gaggacaaca tggcctctct ccctgccact cacgagttgc acatcttcgg ctccatcaac
     5641 ggtgtcgact tcgacatggt cggtcagggc accggcaacc ctaacgacgg ttacgaggag
     5701 ttgaacctta agtccaccaa gggtgacctc cagttctccc cctggattct tgtccctcac
     5761 atcggctacg gcttccacca gtaccttccc taccctgacg gcatgtcccc tttccaggcc
     5821 gccatggttg acggctccgg ttaccaggtc caccgtacta tgcagttcga ggacggtgcc
     5881 tcccttactg ttaactaccg ttacacctac gagggttccc acatcaaggg tgaggcccag
     5941 gtcaagggca ctggtttccc tgctgacggt cctgtcatga ccaactccct taccgctgcc
     6001 gactggtgca ggtccaagaa gacttacccc aacgacaaga ccatcatctc caccttcaag
     6061 tggtcctaca ccactggtaa cggcaagcgt taccgatcca ctgcccgaac cacctacacc
     6121 ttcgccaagc ctatggccgc taactacctt aagaaccagc ccatgtacgt tttccgtaag
     6181 accgagctca agcactccaa gaccgagctc aacttcaagg agtggcagaa ggccttcacc
     6241 gacgtcatgg gcatggacga gctttacaag taaaaggttg agcgcgtaat agtgggttgg
     6301 tcttgtctct atccattcat ctgtgagaca tactgatttc aactttttca tcagataata
     6361 aggatggtgt gagataagca aggctgggtt atgaggtttg ggactagatg gtgtttcgcg
     6421 tctaaaggtg agtcttcgtt ggttcaatca cattgatctg atcgctgatc aattgtcagc
     6481 ctctgaatgt gctcttgaag aaaatggttg tctgtaagaa tccagaacca aaaacggtca
     6541 tttcaagtat gtatcctccg tgaagttcct ttagctcttg ctaatattgg actttagcgt
     6601 gctttagcca aagacagggt cgcgaggtta agaggatggg ccaggaaaaa tctaagttag
     6661 atttgtcaca caaagaaaaa gattgtctca gttttggttt tgtttggttt gatttatgca
     6721 taggaaatat ttatgatcga aatcactcat ttcctgcttc gtaaatatca tctactatat
     6781 gcttcgtaaa tgtccatctc ctacatattg tggatatgat aaatgcaatc ctctcgtcta
     6841 cactctcggc ttatgccttt gtcttctttg cgctcctttt agccttcttt ttattcctag
     6901 tcttcaagcc catcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg
     6961 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     7021 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     7081 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     7141 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     7201 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     7261 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     7321 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     7381 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     7441 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     7501 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     7561 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     7621 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     7681 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     7741 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     7801 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     7861 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     7921 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     7981 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     8041 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     8101 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     8161 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     8221 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     8281 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     8341 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     8401 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     8461 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     8521 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     8581 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     8641 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     8701 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     8761 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     8821 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8881 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8941 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     9001 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     9061 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     9121 gccac
//
