LOCUS       sequence-340161-        5220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRS316-PCnU6-scaffold, contains template for amplification of PCnU6 
            and scaffold for fusion PCR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5220)
  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 5220)
  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: pBHM2329
FEATURES             Location/Qualifiers
     source          1..5220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(8..30)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    19..35
                     /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(43..49,50..67,68..73))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    88..109
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(226..243)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(397..985)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(477..496)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1156..1947,1948..2016))
                     /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     1779..1798
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2017..2121)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    2158..2661
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     2702..2720
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2758..2780)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2880..2899
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2923..3138
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3139..3942
                     /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(4073..4528)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4210..4231)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4422..4441
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4654..4676
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4668..4685
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4669..4685
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4692..4711
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4692..4710
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4743..4759
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4743..4759
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_RNA        5038..5113
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(5131..5147)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5132..5148)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(5176..5194)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(join(5215..5220,1..11))
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(join(5215..5220,1..11))
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
       61 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
      121 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
      181 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
      241 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
      301 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
      361 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
      421 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
      481 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
      541 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
      601 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
      661 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
      721 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
      781 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
      841 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
      901 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
      961 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     1021 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     1081 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     1141 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     1201 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     1261 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     1321 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     1381 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     1441 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     1501 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     1561 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     1621 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     1681 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     1741 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     1801 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     1861 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     1921 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     1981 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     2041 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     2101 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt ccttttcatc
     2161 acgtgctata aaaataatta taatttaaat tttttaatat aaatatataa attaaaaata
     2221 gaaagtaaaa aaagaaatta aagaaaaaat agtttttgtt ttccgaagat gtaaaagact
     2281 ctagggggat cgccaacaaa tactaccttt tatcttgctc ttcctgctct caggtattaa
     2341 tgccgaattg tttcatcttg tctgtgtaga agaccacaca cgaaaatcct gtgattttac
     2401 attttactta tcgttaatcg aatgtatatc tatttaatct gcttttcttg tctaataaat
     2461 atatatgtaa agtacgcttt ttgttgaaat tttttaaacc tttgtttatt tttttttctt
     2521 cattccgtaa ctcttctacc ttctttattt actttctaaa atccaaatac aaaacataaa
     2581 aataaataaa cacagagtaa attcccaaat tattccatca ttaaaagata cgaggcgcgt
     2641 gtaagttaca ggcaagcgat ccgtctaaga aaccattatt atcatgacat taacctataa
     2701 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     2761 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     2821 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     2881 ggcatcagag cagattgtac tgagagtgca ccataccacc ttttcaattc atcatttttt
     2941 ttttattctt ttttttgatt tcggtttcct tgaaattttt ttgattcggt aatctccgaa
     3001 cagaaggaag aacgaaggaa ggagcacaga cttagattgg tatatatacg catatgtagt
     3061 gttgaagaaa catgaaattg cccagtattc ttaacccaac tgcacagaac aaaaacctgc
     3121 aggaaacgaa gataaatcat gtcgaaagct acatataagg aacgtgctgc tactcatcct
     3181 agtcctgttg ctgccaagct atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct
     3241 tcattggatg ttcgtaccac caaggaatta ctggagttag ttgaagcatt aggtcccaaa
     3301 atttgtttac taaaaacaca tgtggatatc ttgactgatt tttccatgga gggcacagtt
     3361 aagccgctaa aggcattatc cgccaagtac aattttttac tcttcgaaga cagaaaattt
     3421 gctgacattg gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa
     3481 tgggcagaca ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag
     3541 caggcggcag aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca
     3601 tgcaagggct ccctatctac tggagaatat actaagggta ctgttgacat tgcgaagagc
     3661 gacaaagatt ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt
     3721 tacgattggt tgattatgac acccggtgtg ggtttagatg acaagggaga cgcattgggt
     3781 caacagtata gaaccgtgga tgatgtggtc tctacaggat ctgacattat tattgttgga
     3841 agaggactat ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta cagaaaagca
     3901 ggctgggaag catatttgag aagatgcggc cagcaaaact aaaaaactgt attataagta
     3961 aatgcatgta tactaaactc acaaattaga gcttcaattt aattatatca gttattaccc
     4021 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta
     4081 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     4141 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     4201 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     4261 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     4321 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     4381 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     4441 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     4501 gcgcttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc aactgttggg
     4561 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     4621 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     4681 ccagtgaatt gtaatacgac tcactatagg gcgaattgga gctccaccgc ggtggcggcc
     4741 gctctagaac tagtggatcc cccttgcatt agaactaaaa acaaagcatg attattacag
     4801 ttcatttatt ttttaaattg atcggcatgc atgcaaagta tacgtgcaag gacaatggta
     4861 acctgcaggt gtgaccgata attataacca tttgttgaga atgaagaggt gaggagaaaa
     4921 acaatggatg acgggaaaaa aataaaaaaa cactgagacg gcgtggaccg ccgtcttatt
     4981 tgcttccgtt atccgccaaa gtggaaattg cacatacacc ggcagggtat actgttggtt
     5041 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     5101 accgagtcgg tgctttttta tcaagcttat cgataccgtc gacctcgagg gggggcccgg
     5161 taccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat catggtcata
//
