LOCUS       sequence-487554-        8025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Linker-Cno_mNeonGreen-CBP-2xFLAG-CnU6-sgRNA-NATR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8025)
  AUTHORS   Nalley MJ, Banerjee S, Huang MY, Madhani HD
  TITLE     Near 100% efficient homology-dependent genome engineering in the 
            human fungal pathogen Cryptococcus neoformans
  JOURNAL   bioRxiv 2025.02.14.638277
REFERENCE   2  (bases 1 to 8025)
  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: "bioRxiv 
            2025.02.14.638277"
COMMENT     Sequence Label: pBHM2660
FEATURES             Location/Qualifiers
     source          1..8025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1058..1074
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1059..1075
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1093..1116
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     regulatory      1381..1390
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1846..1923
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             1936..1983
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     misc_RNA        complement(2323..2398)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(2928..3491)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="TLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFTLR
                     EVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRRLT
                     VEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTLCG
                     LDTALYDGTASDGEQALYMSMPCP"
     primer_bind     complement(4300..4319)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4301..4319)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4326..4343)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4326..4342)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4335..4357)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4483..4938
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4570..4589)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4780..4801
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5069..5872)
                     /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"
     promoter        complement(5873..6088)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6112..6131)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6231..6253
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6291..6309)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6350..6853
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6890..6994
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6995..7063,7064..7855)
                     /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     complement(7213..7232)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctcg aaattaaccc
     1021 tcactaaagg gaacaaaagc tggtaccggg ccccccctcg aggtcgacgg tatcgataag
     1081 cttggtggcg gactcgaagt cttgttccaa ggtcctggat ctggtatggt ctccaagggt
     1141 gaggaggaca acatggcctc cctccccgcc acccacgagc tccacatctt cggttccatc
     1201 aacggtgtcg acttcgacat ggtcggtcag ggtaccggta accccaacga cggttacgag
     1261 gagctcaacc tcaagtccac caagggtgac ctccagttct ccccctggat cctcgtcccc
     1321 cacatcggtt acggtttcca ccagtacctc ccctaccccg acggtatgtc ccccttccag
     1381 gccgccatgg tcgacggttc cggttaccag gtccaccgaa ccatgcagtt cgaggacggt
     1441 gcctccctca ccgtcaacta ccgatacacc tacgagggtt cccacatcaa gggtgaggcc
     1501 caggtcaagg gtaccggttt ccccgccgac ggtcccgtca tgaccaactc cctcaccgcc
     1561 gccgactggt gtcgatccaa gaagacctac cccaacgaca agaccatcat ctccaccttc
     1621 aagtggtcct acaccaccgg taacggtaag cgataccgat ccaccgcccg aaccacctac
     1681 accttcgcca agcccatggc cgccaactac ctcaagaacc agcccatgta cgtcttccga
     1741 aagaccgagc tcaagcactc caagaccgag ctcaacttca aggagtggca gaaggccttc
     1801 accgacgtca tgggtatgga cgagctctac aaggggtcag ggtcaaagag aagatggaaa
     1861 aagaatttca tagccgtctc agcagccaac cgctttaaga aaatctcatc ctccggggca
     1921 cttgggtcag ggtcagatta taaagatgac gatgacaagg attataaaga tgacgatgac
     1981 aagtaatctg gacatgtcaa ttgtacactg ccttataatg ccccatgcac ttccagtatt
     2041 agtcatttga agcgttgaaa gatctcttgg cagtgtgatc atggaaacaa tcccaaacag
     2101 cagcaaactg aaaacgttct ggtatgcaat ccatcccatc gtacaagctc tccaaatacc
     2161 tcttccaatc caaaaacggt ttagatgaaa gtctcgtctg atgcacttgt tagtgacacc
     2221 ccagaatttg atgatcttcg actcacctcg agcggccgcc agtgtgatgg atatctgcag
     2281 aattcgccct tgaatagagt cacttttgct agggataaaa aagcaccgac tcggtgccac
     2341 tttttcaagt tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacct
     2401 aggcatgagg atatctcgca tctagcaaca gtataccctg ccggtgtatg tgcaatttcc
     2461 actttggcgg ataacggaag caaataagac ggcggtccac gccgtctcag tgttttttta
     2521 tttttttccc gtcatccatt gtttttctcc tcacctcttc attctcaaca aatggttata
     2581 attatcggtc acacctgcag gttaccattg tccttgcacg tatactttgc atgcatgccg
     2641 atcaatttaa aaaataaatg aactgtaata atcatgcttt gtttttagtt ctaatgcaag
     2701 ggggatccac tagttgccat gaagatcctg aggattcaac ggcgttgctg agggtgggaa
     2761 ggatggtatt gttgtctgta gatagattaa tgggcatata atgtcaaaaa agatgattga
     2821 cacgatgtct gtctcgaatt cgcagtgtgc taaaattgta tcacttctct aatatgtata
     2881 tatacaccct ctaaggaaaa ttaacccctt accgccttca cgaattctta ggggcagggc
     2941 atgctcatgt agagcgcctg ctcgccgtcc gaggcggtgc cgtcgtacag ggcggtgtcc
     3001 aggccgcaga gggtgaaccc catccgccgg tacgcgtgga tagccggtgc gttaacgttg
     3061 gtgacctcca gccagaggtg cccggcgccc cgctcgcggg cgaactccgt cgcgagcccc
     3121 atcaacgcgc gcccgacccc gtgcccccgg tgctccgggg cgacctcgat gtcctcgacg
     3181 gtcagccggc ggttccagcc ggagtacgag acgaccacga agcccgccag gtcgccgtcg
     3241 tccccgtacg cgacgaacgt ccgggagtcc gggtcgccgt cctccccgtc gtccgattcg
     3301 tcgtccgatt cgtcgtcggg gaacaccttg gtcaggggcg ggtccaccgg cacctcccgc
     3361 agggtgaagc cgtccccggt ggcggtgacg cggaagacgg tgtcggtggt gaaggaccca
     3421 tccagtgcct cgatggcctc ggcgtccccc gggacactgg tgcggtaccg gtaagccgtg
     3481 tcgtcaagag tggcggccgc catagacatg ttgggcgagt ttactaatgg aaaaagacaa
     3541 ttagcaaaga ggctgcatat taggagacgc ggtgaagatt ggggaggaca atagggtccg
     3601 tgcaccaaaa agtcctgacg gcagctctgt tcaacgcaca tccatctctt ccagctatgc
     3661 gtgtggcacc agcacggatg gattgttgca ccaaaaggac aaggcgagat ggatgcaggt
     3721 cgaggcgatg cggtgctatc ggccctcgtt gtcgaacgcc acgtcgtcct ctcggcgacc
     3781 gtcaatcctg gaaggaaatg accaagcgtg cacccagcat ccagcatcca acatccagca
     3841 cccagcacct agcacccatg atccaatggc cggcatgcag atcagcgtgg ggagagcacc
     3901 atctagcagc ccctggcagg cccagcattg tgctcggatg tgcgataggc ggtgggtgcg
     3961 ataaggtgga ttagtggagg atggaggact gagtggcggg caagggaagg gggcgggaag
     4021 atgactcacc ttgggcagtg ggtgggggat gcggtatagg ggatgtatgt ggatgctggc
     4081 ggaggataga agctgtaagc tgagctggtg ccaacggtga cgctgtgaga gtggtctcgg
     4141 cgaccctgac ggcgactcgg cgttaccccg tgtggcacac cggacgcgcg tcgagtgacg
     4201 ttgctgctat tgttgttacc atcatcctct cctccgacaa ccatacactc agtgcacacc
     4261 atgccactcg aatcctgcat gcggtggagc tccaattcgc cctatagtga gtcgtattac
     4321 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4381 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4441 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg
     4501 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4561 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4621 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4681 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4741 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4801 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     4861 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     4921 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4981 acaccgcata gggtaataac tgatataatt aaattgaagc tctaatttgt gagtttagta
     5041 tacatgcatt tacttataat acagtttttt agttttgctg gccgcatctt ctcaaatatg
     5101 cttcccagcc tgcttttctg taacgttcac cctctacctt agcatccctt ccctttgcaa
     5161 atagtcctct tccaacaata ataatgtcag atcctgtaga gaccacatca tccacggttc
     5221 tatactgttg acccaatgcg tctcccttgt catctaaacc cacaccgggt gtcataatca
     5281 accaatcgta accttcatct cttccaccca tgtctctttg agcaataaag ccgataacaa
     5341 aatctttgtc gctcttcgca atgtcaacag tacccttagt atattctcca gtagataggg
     5401 agcccttgca tgacaattct gctaacatca aaaggcctct aggttccttt gttacttctt
     5461 ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa
     5521 tgtctgccca ttctgctatt ctgtatacac ccgcagagta ctgcaatttg actgtattac
     5581 caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct
     5641 ttagcggctt aactgtgccc tccatggaaa aatcagtcaa gatatccaca tgtgttttta
     5701 gtaaacaaat tttgggacct aatgcttcaa ctaactccag taattccttg gtggtacgaa
     5761 catccaatga agcacacaag tttgtttgct tttcgtgcat gatattaaat agcttggcag
     5821 caacaggact aggatgagta gcagcacgtt ccttatatgt agctttcgac atgatttatc
     5881 ttcgtttcct gcaggttttt gttctgtgca gttgggttaa gaatactggg caatttcatg
     5941 tttcttcaac actacatatg cgtatatata ccaatctaag tctgtgctcc ttccttcgtt
     6001 cttccttctg ttcggagatt accgaatcaa aaaaatttca aggaaaccga aatcaaaaaa
     6061 aagaataaaa aaaaaatgat gaattgaaaa ggtggtatgg tgcactctca gtacaatctg
     6121 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     6181 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     6241 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     6301 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgg atcgcttgcc
     6361 tgtaacttac acgcgcctcg tatcttttaa tgatggaata atttgggaat ttactctgtg
     6421 tttatttatt tttatgtttt gtatttggat tttagaaagt aaataaagaa ggtagaagag
     6481 ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa atttcaacaa aaagcgtact
     6541 ttacatatat atttattaga caagaaaagc agattaaata gatatacatt cgattaacga
     6601 taagtaaaat gtaaaatcac aggattttcg tgtgtggtct tctacacaga caagatgaaa
     6661 caattcggca ttaatacctg agagcaggaa gagcaagata aaaggtagta tttgttggcg
     6721 atccccctag agtcttttac atcttcggaa aacaaaaact attttttctt taatttcttt
     6781 ttttactttc tatttttaat ttatatattt atattaaaaa atttaaatta taattatttt
     6841 tatagcacgt gatgaaaagg acccaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6901 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     6961 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     7021 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     7081 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     7141 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     7201 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     7261 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     7321 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     7381 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     7441 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     7501 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     7561 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     7621 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     7681 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     7741 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     7801 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7861 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     7921 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     7981 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
