LOCUS       sequence-478421-        7256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HYGR-CnU6-empty sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7256)
  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 7256)
  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: pBHM2616
FEATURES             Location/Qualifiers
     source          1..7256
                     /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"
     CDS             4544..5560
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="PELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYVL
                     RVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPETE
                     LPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTVM
                     DDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGDS
                     QYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNFD
                     DAAWAQGRCDAIVRSGAGTVGRTQIAHRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     misc_RNA        6089..6164
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(6182..6198)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6183..6199)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(6227..6245)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6266..6282)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6266..6282)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6279..6301)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6290..6306
                     /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(6314..6320,6321..6338,6339..6344))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6359..6380
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6497..6514)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6668..7256)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6748..6767)
                     /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 ttggagctcc accgcatgca ggattcgagt ggcatggtgt gcgctgagtg
     3781 tatggttgtc ggaggagagg atgatggtaa caacaatagc agcaacgtca ctcgacgcgc
     3841 gtccggtgtg ccacacgggg taacgccgag tcgccgtcag ggtcgccgag accactctca
     3901 cagcgtcacc gttggcacca gctcagctta cagcttctat cctccgccag catccacata
     3961 catcccctat accgcatccc ccacccactg cccaaggtga gtcatcttcc cgcccccttc
     4021 ccttgcccgc cactcagtcc tccatcctcc actaatccac cttatcgcac ccaccgccta
     4081 tcgcacatcc gagcacaatg ctgggcctgc caggggctgc tagatggtgc tctccccacg
     4141 ctgatctgca tgccggccat tggatcatgg gtgctaggtg ctgggtgctg gatgttggat
     4201 gctggatgct gggtgcacgc ttggtcattt ccttccagga ttgacggtcg ccgagaggac
     4261 gacgtggcgt tcgacaacga gggccgatag caccgcatcg cctcgacctg catccatctc
     4321 gccttgtcct tttggtgcaa caatccatcc gtgctggtgc cacacgcata gctggaagag
     4381 atggatgtgc gttgaacaga gctgccgtca ggactttttg gtgcacggac cctattgtcc
     4441 tccccaatct tcaccgcgtc tcctaatatg cagcctcttt gctaattgtc tttttccatt
     4501 agtaaactcg cccaacatgt ctatggaaga agaaggtgat ccacctgaac tcaccgcgac
     4561 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
     4621 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     4681 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     4741 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta
     4801 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     4861 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     4921 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     4981 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     5041 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     5101 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     5161 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     5221 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     5281 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     5341 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     5401 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     5461 cgcccacaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     5521 aaaccgacgc cccagcactc gtccgagggc aaaggaatag gaattcgtga ggcggtaagg
     5581 ggttaatttt ccttagaggg tgtatatata catattagag aagtgataca attttagcac
     5641 actgcgaatt cgagacagac atcgtgtcaa tcatcttttt tgacattata tgcccattaa
     5701 tctatctaca gacaacaata ccatccttcc caccctcagc aacgccgttg aatcctcagg
     5761 atcttcatgg caactagtgg atcccccttg cattagaact aaaaacaaag catgattatt
     5821 acagttcatt tattttttaa attgatcggc atgcatgcaa agtatacgtg caaggacaat
     5881 ggtaacctgc aggtgtgacc gataattata accatttgtt gagaatgaag aggtgaggag
     5941 aaaaacaatg gatgacggga aaaaaataaa aaaacactga gacggcgtgg accgccgtct
     6001 tatttgcttc cgttatccgc caaagtggaa attgcacata caccggcagg gtatactgtt
     6061 gctagatgcg agatatcctc atgcctaggt tttagagcta gaaatagcaa gttaaaataa
     6121 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt atcaagctta
     6181 tcgataccgt cgacctcgag ggggggccgg gtaccagctt ttgttccctt tagtgagggt
     6241 taatttcgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     6301 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     6361 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6421 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6481 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     6541 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6601 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6661 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6721 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6781 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6841 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6901 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6961 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     7021 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     7081 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     7141 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     7201 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaa
//
