LOCUS       sequence-480873-        6376 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of DNA into Cryptococcus neoformans Safe Haven 1 locus, 
            contains ptxD marker for selection on media containing phosphite as 
            a sole phosphorus source.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6376)
  AUTHORS   Boucher MJ, Madhani HD
  TITLE     CRISPR/Cas9-compatible plasmids enabling seven dominant genetic 
            selection methods for the human fungal pathogen Cryptococcus 
            neoformans.
  JOURNAL   Microbiol Spectr. 2025 Sep 25:e0193525. doi: 
            10.1128/spectrum.01935-25.
  PUBMED    40996288
REFERENCE   2  (bases 1 to 6376)
  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: "Microbiol 
            Spectr. 2025 Sep 25:e0193525. doi: 10.1128/spectrum.01935-25."
COMMENT     Sequence Label: pBHM2675
FEATURES             Location/Qualifiers
     source          1..6376
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(3845..4210)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     3979..4001
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3993..4010
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3994..4010
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4020..4039
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4020..4038
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    4047..4154
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     4063..4079
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4064..4080
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4114..4130)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4114..4130)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4167..4187)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4167..4185)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4206..4222)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4206..4222)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4219..4241)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4230..4246
                     /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(4254..4260,4261..4278,4279..4284))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4299..4320
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4437..4454)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4608..5196)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4688..4707)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5367..6158,6159..6227))
                     /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     5990..6009
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6228..6332)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agatctgccc atagacttca aatcatggaa ttggacgttt atcttcttgc tttggatatc
       61 caccctatcg tacaatcttg gcactttgga tcatacgagg cactttgcgg actagggaat
      121 ggttatcatc aatttcttgg acctctccat gacacctggc atcgtcgagc taccacagcc
      181 ccctgaattc ttaaaggcga accccccttc ctaggaacct tgagcgctgg actggatggc
      241 actctgagac cgcctatgct taaggacgcg agcttaccct tccattgatg gaagtcgatc
      301 atttatgata atagaacacg gcggaacttg actttacagg ccaagggaag caacaggaga
      361 agaagctaag gttataacag gctcagcata tttgtatata cggtgccatt caacgctgaa
      421 cactttgtac tcacggtctg cccgttgaat aacaccatct gctgatgatc atcccaattt
      481 gtgagtctta tttgccattg atcctggtgt ataaccgata ctgaagccct ttaggtctga
      541 tcagttatag acgtggtata ttcattcttc ctagattagc tatgccgaaa gagatggata
      601 ccttgatgtg actggaatgc catgaagcag gagattgtag cagctcaaat tggagaagga
      661 tgttgcaacc gtcggccgtg aaggtcttgt catatctttg tctgcctggg agaatatgta
      721 caagtgcccc gatgttactg atggaaaaca tcgtgtggcg cgccacgata cttgtgttaa
      781 ttaaaccaaa ccagtctgtg ccaagtggat tgcttatgca tcgagtggcg agcccactga
      841 cctctgggcc ccttttttga actataaaaa gcggccaagt ctgatttcgg tctcacaatc
      901 atcaacatcg acttctcttt tcgcctcgat caacctaaac aacggtcact cagccatgaa
      961 ctggacaatg gaatatttcg ccttgccttg gatctttggc ctctagactc aatcaacgtc
     1021 aggataaagg tgagcatagg ttgctcactc actgtagcct ttttttccct tcttcttggt
     1081 ccagatggtt cccaaggctc tataaagtgt atctgtcagt tccacttcct tactccgaag
     1141 cgattcagag ttgtaaagtg cgtctgaccc cctgattata tctcaaagtc ccttcacaaa
     1201 ttcagacatg atgcgataaa gtgccggatt gtcattcgcg tcttgtgact gtatagaaag
     1261 tgccggcatc ctcttttctt ttcctctttg gccgagcgac tcagagttgt gaagcctgct
     1321 ggtatctgtc tgttcccttg ccctgataaa gaatatcgaa gagcatttct ttgattcaca
     1381 atacgttgac gaatggtgtg atggatccta agcagcgcca gccctttgaa cttgtgtcga
     1441 aaaaaaaaaa aaaacagcat atgatacgta aagggaagaa aaggatggag ctgactgttt
     1501 tgacaggcat aattttgagc gtgcttcatt ggccggttta tctgtattaa cacggaagag
     1561 atgtagaaac tagcttcctg gtttcagaga caaggagcca tgaagatcct gaggattcaa
     1621 cggcgttgct gagggtggga aggatggtat tgttgtctgt agatagatta atgggcatat
     1681 aatgtcaaaa aagatgattg acacgatgtc tgtctcgaat tcgcagtgtg ctaaaattgt
     1741 atcacttctc taatatgtat atatacaccc tctaaggaaa attaacccct taccgccttc
     1801 acgaattctt aacaggcggc gggctcggcc ttggggagtc ggttggcggc gttgatgggt
     1861 cgggcaccgg cgaggacctg gatgatgttc tgggcggcac atcgctcgat ctcgagtcgg
     1921 acggctcgga cggcggaacc gatgtggggg gtgaagaggg tgttggggtg ggcgaggagg
     1981 gcggggtcga tgagtcgggg tcggtcggct cgggcccagt cctccatctc gaagacgtcg
     2041 gcggcgtaac caccgagctg acctcgctcg agggcggcga ggacggcggc ctcgtcgacg
     2101 acggaacctc gacaggggtt gacgaggagg gcaccgggtc ggacgagggc gaggagctcg
     2161 gcgttgacga ggtgctgggt gtcggcgttg agggggaggg cgaggaggat gaagtcggag
     2221 gaggcgaaga gctcggaaca ggcgacctgt cggagaccga gtcgctgctc ggtctgggtg
     2281 tcgagggcct tggcctcgtg gtactggagg gtggcacccc aaccctggag tcggtcggcc
     2341 atggcaagac ctatggcacc cataccgagg ataccgacgg tggcgttgtc gagaccggta
     2401 ccgtagaact ggggctgcca accctggaac tcaccggatc ggacgaaggc gtcggcggct
     2461 cggaggtgtc gaccgagacc ctgtcgggag atgttagtgg agtaacatgt tgcgggaaac
     2521 ggggttacat acgacggcga gcccgatagc gagctcggcg gtggggacgg tgaggaggtc
     2581 ggggacgaag gtgagccaga cacctcgggc ggtgcaggca tcgacgtcga agttgtcgaa
     2641 acccttgagg gcacaaccga cgactcggag ctcgggacag gcctggagga agtcggcgtc
     2701 gactcggtcg ggcatgaagg ccatcatggc ctgggcgtct cgacatcgtc ggaggatctc
     2761 ctctcgggtg agggtggagt cggtctggtt ggtcatgagc tcacagtggg gggcgaggag
     2821 ctggaggatc tcgtcgtgga ctcggtgggt gatgacgagc ttggggagca tggcggccgc
     2881 catagacatg ttgggcgagt ttactaatgg aaaaagacaa ttagcaaaga ggctgcatat
     2941 taggagacgc ggtgaagatt ggggaggaca atagggtccg tgcaccaaaa agtcctgacg
     3001 gcagctctgt tcaacgcaca tccatctctt ccagctatgc gtgtggcacc agcacggatg
     3061 gattgttgca ccaaaaggac aaggcgagat ggatgcaggt cgaggcgatg cggtgctatc
     3121 ggccctcgtt gtcgaacgcc acgtcgtcct ctcggcgacc gtcaatcctg gaaggaaatg
     3181 accaagcgtg cacccagcat ccagcatcca acatccagca cccagcacct agcacccatg
     3241 atccaatggc cggcatgcag atcagcgtgg ggagagcacc atctagcagc ccctggcagg
     3301 cccagcattg tgctcggatg tgcgataggc ggtgggtgcg ataaggtgga ttagtggagg
     3361 atggaggact gagtggcggg caagggaagg gggcgggaag atgactcacc ttgggcagtg
     3421 ggtgggggat gcggtatagg ggatgtatgt ggatgctggc ggaggataga agctgtaagc
     3481 tgagctggtg ccaacggtga cgctgtgaga gtggtctcgg cgaccctgac ggcgactcgg
     3541 cgttaccccg tgtggcacac cggacgcgcg tcgagtgacg ttgctgctat tgttgttacc
     3601 atcatcctct cctccgacaa ccatacactc agtgcacacc atgccactcg aatcctgcat
     3661 gcttatgtga gtcctcccct tcccgtgcgc cgctctccag ctcacatcct cgcagccatg
     3721 gactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3781 attggttaaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     3841 acgcttacaa tttccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     3901 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     3961 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt
     4021 aatacgactc actatagggc gaattgggta ccgggccccc cctcgaggtc gacggtatcg
     4081 ataagcttga tatcgaattc ctgcagcccg ggggatccac tagttctaga gcggccgcca
     4141 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     4201 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4261 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4321 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4381 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4441 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4501 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4561 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4621 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4681 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4741 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4801 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4861 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4921 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4981 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5041 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5101 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5161 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5221 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5281 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5341 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5401 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5461 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5521 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5581 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5641 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5701 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5761 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5821 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5881 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5941 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6001 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6061 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6121 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6181 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6241 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6301 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6361 gcgccctgta gcggca
//
