LOCUS       sequence-480872-        5788 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of DNA into Cryptococcus neoformans Safe Haven 1 locus, 
            contains BSR marker for selection on blasticidin S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5788)
  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 5788)
  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: pBHM2674
FEATURES             Location/Qualifiers
     source          1..5788
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(3257..3622)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     3391..3413
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3405..3422
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3406..3422
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3432..3451
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3432..3450
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    3459..3566
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     3475..3491
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3476..3492
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3526..3542)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3526..3542)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3579..3599)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3579..3597)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3618..3634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3618..3634)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3631..3653)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3642..3658
                     /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(3666..3672,3673..3690,3691..3696))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3711..3732
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3849..3866)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4020..4608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4100..4119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4779..5570,5571..5639))
                     /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     5402..5421
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5640..5744)
                     /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 agtttcgggt gtacttgagg gggatgagct cctcgatggt ggtcttgacg
     1861 agcttaccgt tcatctcgat gaggacgaaa cagtcggggg cgtagtcgga gatgagctct
     1921 cgacacatac cacaggggga gacgactcgg atggatcggt cgacctcgtc ggagtagggg
     1981 tgtcggacgg cgacgatggt gtcgaagtcc ttctgaccgt tggagacggc ggaaccgatg
     2041 gcgatggcct cggcacagac ggtgactcga ccgatgtagg cctcgatgtg ctgtcgggag
     2101 atgttagtgg agtaacatgt tgcgggaaac ggggttacat acgacggcgg agatgatctc
     2161 accggtcttg gttcggatgg cggcaccgac gtggtgcttg ttgtcctcgt agagcatggt
     2221 gatcttctcg gtggcgacct cgacgagctc gaggtcctgc tgggagatgt tgaaggtctt
     2281 catggcggcc gccatagaca tgttgggcga gtttactaat ggaaaaagac aattagcaaa
     2341 gaggctgcat attaggagac gcggtgaaga ttggggagga caatagggtc cgtgcaccaa
     2401 aaagtcctga cggcagctct gttcaacgca catccatctc ttccagctat gcgtgtggca
     2461 ccagcacgga tggattgttg caccaaaagg acaaggcgag atggatgcag gtcgaggcga
     2521 tgcggtgcta tcggccctcg ttgtcgaacg ccacgtcgtc ctctcggcga ccgtcaatcc
     2581 tggaaggaaa tgaccaagcg tgcacccagc atccagcatc caacatccag cacccagcac
     2641 ctagcaccca tgatccaatg gccggcatgc agatcagcgt ggggagagca ccatctagca
     2701 gcccctggca ggcccagcat tgtgctcgga tgtgcgatag gcggtgggtg cgataaggtg
     2761 gattagtgga ggatggagga ctgagtggcg ggcaagggaa gggggcggga agatgactca
     2821 ccttgggcag tgggtggggg atgcggtata ggggatgtat gtggatgctg gcggaggata
     2881 gaagctgtaa gctgagctgg tgccaacggt gacgctgtga gagtggtctc ggcgaccctg
     2941 acggcgactc ggcgttaccc cgtgtggcac accggacgcg cgtcgagtga cgttgctgct
     3001 attgttgtta ccatcatcct ctcctccgac aaccatacac tcagtgcaca ccatgccact
     3061 cgaatcctgc atgcttatgt gagtcctccc cttcccgtgc gccgctctcc agctcacatc
     3121 ctcgcagcca tggactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3181 cgatttcggc ctattggtta aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3241 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
     3301 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     3361 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     3421 gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagg
     3481 tcgacggtat cgataagctt gatatcgaat tcctgcagcc cgggggatcc actagttcta
     3541 gagcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg
     3601 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3661 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3721 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3781 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3841 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3901 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3961 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4021 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4081 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4141 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4201 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4261 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4321 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4381 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4441 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4501 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4561 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4621 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4681 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4741 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4801 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4861 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4921 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4981 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5041 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5101 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5161 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5221 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5281 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5341 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5401 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5461 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5521 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5581 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5641 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5701 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5761 gtgccacctg acgcgccctg tagcggca
//
