LOCUS       sequence-341226-        7730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRS316-mNeonGreen-CBP-2XFLAG-NAT1, contains C. neoformans optimized 
            mNeonGreen and NAT1 marker for use as PCR template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7730)
  AUTHORS   Huang MY, Joshi MB, Boucher MJ, Lee S, Loza LC, Gaylord EA, Doering 
            TL, Madhani HD
  TITLE     Short homology-directed repair using optimized Cas9 in the pathogen 
            Cryptococcus neoformans enables rapid gene deletion and tagging.
  JOURNAL   Genetics. 2021 Oct 23. pii: 6409193. doi: 10.1093/genetics/iyab180.
  PUBMED    34791226
REFERENCE   2  (bases 1 to 7730)
  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: "Genetics. 
            2021 Oct 23. pii: 6409193. doi: 10.1093/genetics/iyab180."
COMMENT     Sequence Label: pBHM2404
FEATURES             Location/Qualifiers
     source          1..7730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /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(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1939..1956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1940..1956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1963..1982
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1963..1981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2007..2030
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     regulatory      2295..2304
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2760..2837
                     /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             2850..2897
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4072..4635
                     /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(4912..4928)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4913..4929)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(4957..4975)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4996..5012)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4996..5012)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5009..5031)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5020..5036
                     /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(5044..5050,5051..5068,5069..5074))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5089..5110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5227..5244)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5398..5986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5478..5497)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6157..6948,6949..7017))
                     /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     6780..6799
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7018..7122)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7159..7662
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     7703..7721
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     1921 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
     1981 ggcgaattgg agctccaggt ggcggactcg aagtcttgtt ccaaggtcct ggatctggta
     2041 tggtctccaa gggtgaggag gacaacatgg cctccctccc cgccacccac gagctccaca
     2101 tcttcggttc catcaacggt gtcgacttcg acatggtcgg tcagggtacc ggtaacccca
     2161 acgacggtta cgaggagctc aacctcaagt ccaccaaggg tgacctccag ttctccccct
     2221 ggatcctcgt cccccacatc ggttacggtt tccaccagta cctcccctac cccgacggta
     2281 tgtccccctt ccaggccgcc atggtcgacg gttccggtta ccaggtccac cgaaccatgc
     2341 agttcgagga cggtgcctcc ctcaccgtca actaccgata cacctacgag ggttcccaca
     2401 tcaagggtga ggcccaggtc aagggtaccg gtttccccgc cgacggtccc gtcatgacca
     2461 actccctcac cgccgccgac tggtgtcgat ccaagaagac ctaccccaac gacaagacca
     2521 tcatctccac cttcaagtgg tcctacacca ccggtaacgg taagcgatac cgatccaccg
     2581 cccgaaccac ctacaccttc gccaagccca tggccgccaa ctacctcaag aaccagccca
     2641 tgtacgtctt ccgaaagacc gagctcaagc actccaagac cgagctcaac ttcaaggagt
     2701 ggcagaaggc cttcaccgac gtcatgggta tggacgagct ctacaagggg tcagggtcaa
     2761 agagaagatg gaaaaagaat ttcatagccg tctcagcagc caaccgcttt aagaaaatct
     2821 catcctccgg ggcacttggg tcagggtcag attataaaga tgacgatgac aaggattata
     2881 aagatgacga tgacaagtaa tctggacatg tcaattgtac actgccttat aatgccccat
     2941 gcacttccag tattagtcat ttgaagcgtt gaaagatctc ttggcagtgt gatcatggaa
     3001 acaatcccaa acagcagcaa actgaaaacg ttctggtatg caatccatcc catcgtacaa
     3061 gctctccaaa tacctcttcc aatccaaaaa cggtttagat gaaagtctcg tctgatgcac
     3121 ttgttagtga caccccagaa tttgatgatc ttcgactcac ctcgagcggc cgccagtgtg
     3181 atggatatct gcagaattcg cccttgaata gagtcacttt tgctaggctg cgaggatgtg
     3241 agctggagag cggcgcacgg gaaggggagg actcacataa gcatgcagga ttcgagtggc
     3301 atggtgtgca ctgagtgtat ggttgtcgga ggagaggatg atggtaacaa caatagcagc
     3361 aacgtcactc gacgcgcgtc cggtgtgcca cacggggtaa cgccgagtcg ccgtcagggt
     3421 cgccgagacc actctcacag cgtcaccgtt ggcaccagct cagcttacag cttctatcct
     3481 ccgccagcat ccacatacat cccctatacc gcatccccca cccactgccc aaggtgagtc
     3541 atcttcccgc ccccttccct tgcccgccac tcagtcctcc atcctccact aatccacctt
     3601 atcgcaccca ccgcctatcg cacatccgag cacaatgctg ggcctgccag gggctgctag
     3661 atggtgctct ccccacgctg atctgcatgc cggccattgg atcatgggtg ctaggtgctg
     3721 ggtgctggat gttggatgct ggatgctggg tgcacgcttg gtcatttcct tccaggattg
     3781 acggtcgccg agaggacgac gtggcgttcg acaacgaggg ccgatagcac cgcatcgcct
     3841 cgacctgcat ccatctcgcc ttgtcctttt ggtgcaacaa tccatccgtg ctggtgccac
     3901 acgcatagct ggaagagatg gatgtgcgtt gaacagagct gccgtcagga ctttttggtg
     3961 cacggaccct attgtcctcc ccaatcttca ccgcgtctcc taatatgcag cctctttgct
     4021 aattgtcttt ttccattagt aaactcgccc aacatgtcta tggcggccgc cactcttgac
     4081 gacacggctt accggtaccg caccagtgtc ccgggggacg ccgaggccat cgaggcactg
     4141 gatgggtcct tcaccaccga caccgtcttc cgcgtcaccg ccaccgggga cggcttcacc
     4201 ctgcgggagg tgccggtgga cccgcccctg accaaggtgt tccccgacga cgaatcggac
     4261 gacgaatcgg acgacgggga ggacggcgac ccggactccc ggacgttcgt cgcgtacggg
     4321 gacgacggcg acctggcggg cttcgtggtc gtctcgtact ccggctggaa ccgccggctg
     4381 accgtcgagg acatcgaggt cgccccggag caccgggggc acggggtcgg gcgcgcgttg
     4441 atggggctcg cgacggagtt cgcccgcgag cggggcgccg ggcacctctg gctggaggtc
     4501 accaacgtta acgcaccggc tatccacgcg taccggcgga tggggttcac cctctgcggc
     4561 ctggacaccg ccctgtacga cggcaccgcc tcggacggcg agcaggcgct ctacatgagc
     4621 atgccctgcc cctaagaatt cgtgaaggcg gtaaggggtt aattttcctt agagggtgta
     4681 tatatacata ttagagaagt gatacaattt tagcacactg cgaattcgag acagacatcg
     4741 tgtcaatcat cttttttgac attatatgcc cattaatcta tctacagaca acaataccat
     4801 ccttcccacc ctcagcaacg ccgttgaatc ctcaggatct tcatggctcc ttgtctctga
     4861 aaccaggaag ctagtttcta catctcttcc gtgttaatac agataaacca tcgataccgt
     4921 cgacctcgag ggggggcccg gtaccagctt ttgttccctt tagtgagggt taatttcgag
     4981 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5041 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5101 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5161 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5221 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5281 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5341 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5401 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5461 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5521 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5581 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5641 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5701 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5761 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5821 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5881 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5941 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6001 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6061 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6121 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6181 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6241 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6301 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6361 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6421 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6481 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6541 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6601 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6661 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6721 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6781 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6841 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6901 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6961 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7021 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7081 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7141 gccacctggg tccttttcat cacgtgctat aaaaataatt ataatttaaa ttttttaata
     7201 taaatatata aattaaaaat agaaagtaaa aaaagaaatt aaagaaaaaa tagtttttgt
     7261 tttccgaaga tgtaaaagac tctaggggga tcgccaacaa atactacctt ttatcttgct
     7321 cttcctgctc tcaggtatta atgccgaatt gtttcatctt gtctgtgtag aagaccacac
     7381 acgaaaatcc tgtgatttta cattttactt atcgttaatc gaatgtatat ctatttaatc
     7441 tgcttttctt gtctaataaa tatatatgta aagtacgctt tttgttgaaa ttttttaaac
     7501 ctttgtttat ttttttttct tcattccgta actcttctac cttctttatt tactttctaa
     7561 aatccaaata caaaacataa aaataaataa acacagagta aattcccaaa ttattccatc
     7621 attaaaagat acgaggcgcg tgtaagttac aggcaagcga tccgtctaag aaaccattat
     7681 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
