LOCUS       sequence-479861-        6695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construction of fused marker-sgRNA inserts for Cryptococcus 
            neoformans genome editing, contains BLE marker for selection on 
            phleomycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6695)
  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 6695)
  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: pBHM2678
FEATURES             Location/Qualifiers
     source          1..6695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    1173..1676
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     1717..1735
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1773..1795)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1895..1914
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1938..2153
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             2154..2957
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(3088..3543)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3225..3246)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3437..3456
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3669..3691
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3683..3700
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3684..3700
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3707..3726
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3707..3725
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4525..4534
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        5528..5603
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(5621..5637)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5622..5638)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(5666..5684)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5705..5721)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5705..5721)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5718..5740)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5729..5745
                     /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(5753..5759,5760..5777,5778..5783))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5798..5819
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5936..5953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6107..6695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6187..6206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgggtccttt tcatcacgtg ctataaaaat aattataatt
     1201 taaatttttt aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa
     1261 aaaatagttt ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta
     1321 ccttttatct tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt
     1381 gtagaagacc acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt
     1441 atatctattt aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt
     1501 gaaatttttt aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt
     1561 tatttacttt ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc
     1621 caaattattc catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc
     1681 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     1741 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     1801 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     1861 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     1921 gtgcaccata ccaccttttc aattcatcat ttttttttta ttcttttttt tgatttcggt
     1981 ttccttgaaa tttttttgat tcggtaatct ccgaacagaa ggaagaacga aggaaggagc
     2041 acagacttag attggtatat atacgcatat gtagtgttga agaaacatga aattgcccag
     2101 tattcttaac ccaactgcac agaacaaaaa cctgcaggaa acgaagataa atcatgtcga
     2161 aagctacata taaggaacgt gctgctactc atcctagtcc tgttgctgcc aagctattta
     2221 atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt ggatgttcgt accaccaagg
     2281 aattactgga gttagttgaa gcattaggtc ccaaaatttg tttactaaaa acacatgtgg
     2341 atatcttgac tgatttttcc atggagggca cagttaagcc gctaaaggca ttatccgcca
     2401 agtacaattt tttactcttc gaagacagaa aatttgctga cattggtaat acagtcaaat
     2461 tgcagtactc tgcgggtgta tacagaatag cagaatgggc agacattacg aatgcacacg
     2521 gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc ggcagaagaa gtaacaaagg
     2581 aacctagagg ccttttgatg ttagcagaat tgtcatgcaa gggctcccta tctactggag
     2641 aatatactaa gggtactgtt gacattgcga agagcgacaa agattttgtt atcggcttta
     2701 ttgctcaaag agacatgggt ggaagagatg aaggttacga ttggttgatt atgacacccg
     2761 gtgtgggttt agatgacaag ggagacgcat tgggtcaaca gtatagaacc gtggatgatg
     2821 tggtctctac aggatctgac attattattg ttggaagagg actatttgca aagggaaggg
     2881 atgctaaggt agagggtgaa cgttacagaa aagcaggctg ggaagcatat ttgagaagat
     2941 gcggccagca aaactaaaaa actgtattat aagtaaatgc atgtatacta aactcacaaa
     3001 ttagagcttc aatttaatta tatcagttat taccctatgc ggtgtgaaat accgcacaga
     3061 tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg
     3121 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     3181 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     3241 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     3301 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     3361 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     3421 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     3481 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     3541 cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     3601 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     3661 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact
     3721 atagggcgaa ttggagctcg catgcaggat tcgagtggca tggtgtgcac tgagtgtatg
     3781 gttgtcggag gagaggatga tggtaacaac aatagcagca acgtcactcg acgcgcgtcc
     3841 ggtgtgccac acggggtaac gccgagtcgc cgtcagggtc gccgagacca ctctcacagc
     3901 gtcaccgttg gcaccagctc agcttacagc ttctatcctc cgccagcatc cacatacatc
     3961 ccctataccg catcccccac ccactgccca aggtgagtca tcttcccgcc cccttccctt
     4021 gcccgccact cagtcctcca tcctccacta atccacctta tcgcacccac cgcctatcgc
     4081 acatccgagc acaatgctgg gcctgccagg ggctgctaga tggtgctctc cccacgctga
     4141 tctgcatgcc ggccattgga tcatgggtgc taggtgctgg gtgctggatg ttggatgctg
     4201 gatgctgggt gcacgcttgg tcatttcctt ccaggattga cggtcgccga gaggacgacg
     4261 tggcgttcga caacgagggc cgatagcacc gcatcgcctc gacctgcatc catctcgcct
     4321 tgtccttttg gtgcaacaat ccatccgtgc tggtgccaca cgcatagctg gaagagatgg
     4381 atgtgcgttg aacagagctg ccgtcaggac tttttggtgc acggacccta ttgtcctccc
     4441 caatcttcac cgcgtctcct aatatgcagc ctctttgcta attgtctttt tccattagta
     4501 aactcgccca acatgtctat ggcggccgcc atggccaagc tcacctccgc cgtccccgtc
     4561 ctcaccgccc gagacgtcgc cggtgccgtc gagttctgga ccgaccgact cggtttctcc
     4621 cgagacttcg tcgaggacga cttcgccggt gtcgtcgtat gtaaccccgt ttcccgcaac
     4681 atgttactcc actaacatct cccgacagcg agacgacgtc accctcttca tctccgccgt
     4741 ccaggaccag gtcgtccccg acaacaccct cgcctgggtc tgggtccgag gtctcgacga
     4801 gctctacgcc gagtggtccg aggtcgtctc caccaacttc cgagacgcct ccggtcccgc
     4861 catgaccgag atcggtgagc agccctgggg tcgagagttc gccctccgag accccgccgg
     4921 taactgtgtc cacttcgtcg ccgaggagca ggactaagaa ttcgtgaagg cggtaagggg
     4981 ttaattttcc ttagagggtg tatatataca tattagagaa gtgatacaat tttagcacac
     5041 tgcgaattcg agacagacat cgtgtcaatc atcttttttg acattatatg cccattaatc
     5101 tatctacaga caacaatacc atccttccca ccctcagcaa cgccgttgaa tcctcaggat
     5161 cttcatggct ccttgtctct gaaaccagga agctagtttc tacatctctt cactagtgga
     5221 tcccccttgc attagaacta aaaacaaagc atgattatta cagttcattt attttttaaa
     5281 ttgatcggca tgcatgcaaa gtatacgtgc aaggacaatg gtaacctgca ggtgtgaccg
     5341 ataattataa ccatttgttg agaatgaaga ggtgaggaga aaaacaatgg atgacgggaa
     5401 aaaaataaaa aaacactgag acggcgtgga ccgccgtctt atttgcttcc gttatccgcc
     5461 aaagtggaaa ttgcacatac accggcaggg tatactgttg ctagatgcga gatatcctca
     5521 tgcctaggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     5581 aaaaagtggc accgagtcgg tgctttttta tcaagcttat cgataccgtc gacctcgagg
     5641 gggggccggg taccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat
     5701 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5761 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5821 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5881 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5941 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     6001 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     6061 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     6121 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6181 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6241 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6301 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6361 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6421 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6481 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6541 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6601 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6661 agcagcagat tacgcgcaga aaaaaaggat ctcaa
//
