LOCUS       sequence-443446-        9128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoterless (SwaI Cutsite) amdS:mRuby3 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: pLSPR22
FEATURES             Location/Qualifiers
     source          1..9128
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1820..2620)
                     /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(1931..1950)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2541..2560
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3710..3732
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3724..3741
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3725..3741
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3751..3770
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3751..3769
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     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 cgcgccctgt agcggcaaga tctgcccata gacttcaaat catggaattg gacgtttatc
       61 ttcttgcttt ggatatccac cctatcgtac aatcttggca ctttggatca tacgaggcac
      121 tttgcggact agggaatggt tatcatcaat ttcttggacc tctccatgac acctggcatc
      181 gtcgagctac cacagccccc tgaattctta aaggcgaacc ccccttccta ggaaccttga
      241 gcgctggact ggatggcact ctgagaccgc ctatgcttaa ggacgcgagc ttacccttcc
      301 attgatggaa gtcgatcatt tatgataata gaacacggcg gaacttgact ttacaggcca
      361 agggaagcaa caggagaaga agctaaggtt ataacaggct cagcatattt gtatatacgg
      421 tgccattcaa cgctgaacac tttgtactca cggtctgccc gttgaataac accatctgct
      481 gatgatcatc ccaatttgtg agtcttattt gccattgatc ctggtgtata accgatactg
      541 aagcccttta ggtctgatca gttatagacg tggtatattc attcttccta gattagctat
      601 gccgaaagag atggatacct tgatgtgact ggaatgccat gaagcaggag attgtagcag
      661 ctcaaattgg agaaggatgt tgcaaccgtc ggccgtgaag gtcttgtcat atctttgtct
      721 gcctgggaga atatgtacaa gtgccccgat gttactgatg gaaaacatcg tgtggcgcgc
      781 cacgatactt gtgttaatta aaccaaacca gtctgtgcca agtggattgc ttatgcatcg
      841 agtggcgagc ccactgacct ctgggcccct tttttgaact ataaaaagcg gccaagtctg
      901 atttcggtct cacaatcatc aacatcgact tctcttttcg cctcgatcaa cctaaacaac
      961 ggtcactcag ccatgaactg gacaatggaa tatttcgcct tgccttggat ctttggcctc
     1021 tagactcaat caacgtcagg ataaaggtga gcataggttg ctcactcact gtagcctttt
     1081 tttcccttct tcttggtcca gatggttccc aaggctctat aaagtgtatc tgtcagttcc
     1141 acttccttac tccgaagcga ttcagagttg taaagtgcgt ctgaccccct gattatatct
     1201 caaagtccct tcacaaattc agacatgatg cgataaagtg ccggattgtc attcgcgtct
     1261 tgtgactgta tagaaagtgc cggcatcctc ttttcttttc ctctttggcc gagcgactca
     1321 gagttgtgaa gcctgctggt atctgtctgt tcccttgccc tgataaagaa tatcgaagag
     1381 catttctttg attcacaata cgttgacgaa tggtgtgatg gatcctaagc agcgccagcc
     1441 ctttgaactt gtgtcgaaaa aaaaaaaaaa acagcatatg atacgtaaag ggaagaaaag
     1501 gatggagctg actgttttga caggcataat tttgagcgtg cttcattggc cggtttatct
     1561 gtattaacac ggaagagatg tagaaactag cttcctggtt tcagagacaa ggagccatga
     1621 agatcctgag gattcaacgg cgttgctgag ggtgggaagg atggtattgt tgtctgtaga
     1681 tagattaatg ggcatataat gtcaaaaaag atgattgaca cgatgtctgt ctcgaattcg
     1741 cagtgtgcta aaattgtatc acttctctaa tatgtatata tacaccctct aaggaaaatt
     1801 aaccccttac cgccttcact cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
     1861 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa
     1921 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     1981 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     2041 aggcatcgcc atgggtcacg acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg
     2101 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     2161 gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg
     2221 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     2281 tctcggcagg agcaaggtgg gatgacagga gatcctgccc cggcacttcg cccaatagca
     2341 gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg
     2401 tggccagcca cgatagccgc gctgcctcgt cctgcagttc attcagggca ccggacaggt
     2461 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag
     2521 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg
     2581 gagaacctgc gtgcaatcca tcttgttcaa tcatagacat gttgggcgag tttactaatg
     2641 gaaaaagaca attagcaaag aggctgcata ttaggagacg cggtgaagat tggggaggac
     2701 aatagggtcc gtgcaccaaa aagtcctgac ggcagctctg ttcaacgcac atccatctct
     2761 tccagctatg cgtgtggcac cagcacggat ggattgttgc accaaaagga caaggcgaga
     2821 tggatgcagg tcgaggcgat gcggtgctat cggccctcgt tgtcgaacgc cacgtcgtcc
     2881 tctcggcgac cgtcaatcct ggaaggaaat gaccaagcgt gcacccagca tccagcatcc
     2941 aacatccagc acccagcacc tagcacccat gatccaatgg ccggcatgca gatcagcgtg
     3001 gggagagcac catctagcag cccctggcag gcccagcatt gtgctcggat gtgcgatagg
     3061 cggtgggtgc gataaggtgg attagtggag gatggaggac tgagtggcgg gcaagggaag
     3121 ggggcgggaa gatgactcac cttgggcagt gggtggggga tgcggtatag gggatgtatg
     3181 tggatgctgg cggaggatag aagctgtaag ctgagctggt gccaacggtg acgctgtgag
     3241 agtggtctcg gcgaccctga cggcgactcg gcgttacccc gtgtggcaca ccggacgcgc
     3301 gtcgagtgac gttgctgcta ttgttgttac catcatcctc tcctccgaca accatacact
     3361 cagtgcacac catgccactc gaatcctgca tgcttatgtg agtcctcccc ttcccgtgcg
     3421 ccgctctcca gctcacatcc tcgcagccat ggactcaacc ctatctcggt ctattctttt
     3481 gatttataag ggattttgcc gatttcggcc tattggttaa aaaaatgagc tgatttaaca
     3541 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc gccattcagg
     3601 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     3661 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     3721 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt
     3781 accgggcccc ccctcgaatt taaatatgcc tcagtcctgg gaggagctcg ccgctgacaa
     3841 gcgtgcccgt ctcgctaaga ccatccctga cgagtggaag gtccagaccc tccctgccga
     3901 ggactccgtt attgacttcc ctaagaagtc tggcatcctt tctgaggccg agctcaagat
     3961 cactgaggcc tccgctgctg accttgtctc caagctcgcc gccggtgagt tgacctctgt
     4021 cgaggttacc cttgctttct gtaagcgagc tgctatcgcc cagcagttga ctaactgcgc
     4081 ccacgagttc ttccctgacg ccgctctcgc ccaggctagg gagctcgacg agtactacgc
     4141 taagcacaag aggcccgttg gtcctctcca cggcctcccc atctctctca aggaccagct
     4201 tcgagtcaag ggctacgaga cttctatggg ctacatctct tggcttaaca agtacgacga
     4261 gggcgactct gttctcacta ccatgctccg taaggccggt gccgtcttct acgtcaagac
     4321 ctctgtcccc cagaccctca tggtctgcga gactgtcaac aacatcatcg gccgtaccgt
     4381 caaccctcgt aacaagaact ggtcttgcgg cggctcctct ggtggtgagg gtgccatcgt
     4441 tggcattcgt ggtggcgtca tcggtgttgg taccgacatt ggtggctcta ttcgagtccc
     4501 cgccgccttc aacttcctct acggtcttag gccctcccac ggccgactcc cctacgctaa
     4561 gatggccaac tccatggagg gtcaggagac cgtccactcc gttgtcggcc ccattaccca
     4621 ctctgttgag gacctccgtc tcttcaccaa gtccgtcctc ggtcaggagc cttggaagta
     4681 cgactccaag gtcatcccca tgccctggcg tcagtccgag tctgacatta ttgcctccaa
     4741 gatcaagaac ggcggcctca acatcggcta ctacaacttc gacggcaacg tccttcctca
     4801 ccctcctatc ctccgtggcg tcgagaccac cgtcgccgct ctcgccaagg ccggtcacac
     4861 cgtcaccccc tggacccctt acaagcacga cttcggccac gacctcatct cccacatcta
     4921 cgccgctgac ggctccgccg acgttatgcg tgacatctcc gcttccggcg agcccgccat
     4981 tcctaacatc aaggaccttc tcaaccccaa catcaaggct gttaacatga acgagctctg
     5041 ggacacccac ctccagaagt ggaactacca gatggagtac cttgagaagt ggcgagaggc
     5101 tgaggagaag gccggcaagg aggtgagtag atcacaattt gtaacaggtt ggatgataag
     5161 tccccggtct aacatgtagc gacgacactg aagctcactt cttgtagctc gacgccatca
     5221 tcgcccccat tacccctacc gctgccgttc gacacgacca gttccgatac tacggctacg
     5281 cctctgtcat caacctcctc gacttcacct ccgtcgttgt tcccgttacc ttcgccgaca
     5341 agaacatcga caagaagaac gagtccttca aggccgtttc cgagcttgac gccctcgtcc
     5401 aggaggagta cgaccccgag gcctaccacg gcgctcccgt tgctgtccag gttatcggtc
     5461 gaaggctctc cgaggagagg accttggcca ttgctgagga ggtcggcaag ttgctcggta
     5521 acgtcgtcac tcctggttct ggcggtggtg gctccggcat ggtctctaag ggcgaggagc
     5581 tcatcaagga gaacatgcgt atgaaggtcg tcatggaggg ttctgtcaac ggccaccagt
     5641 tcaagtgcac tggtgagggt gagggcaggc cctacgaggg tgtccagacc atgaggatca
     5701 aggtcatcga gggtggtccc ctccctttcg ccttcgacat tcttgccact tctttcatgt
     5761 acggctcccg taccttcatc aagtaccccg ccgacatccc tgacttcttc aagcagtcct
     5821 tccctgaggg tttcacttgg gagagggtta ccaggtacga ggacggtggt gtcgtcaccg
     5881 tcacccagga cacctccctt gaggacggcg agctcgtcta caacgtcaag gtcaggggcg
     5941 ttaacttccc ctccaacggt cccgtcatgc agaagaagac caagggttgg gagcctaaca
     6001 ctgagatgat gtaccctgct gacggtggtc tcaggggtta cactgacatc gctctcaagg
     6061 ttgacggtgg tggccacctc cactgcaact tcgtcactac ttacaggtct aagaagaccg
     6121 tcggcaacat caagatgccc ggtgtccacg ccgttgacca ccgtctcgag aggatcgagg
     6181 agtccgacaa cgagacctac gttgtccaga gggaggtcgc tgttgccaag tactccaacc
     6241 ttggtggtgg catggacgag ctctacaagt 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 ccacctga
//
