LOCUS       sequence-254590-        7190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA ribozyme construct with Cryptococcus neoformans ACT1 promter 
            and TRP1 terminator. Following ribozyme cleavage, a gRNA targeting 
            ADE2 is liberated..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7190)
  AUTHORS   Arras SD, Chua SM, Wizrah MS, Faint JA, Yap AS, Fraser JA
  TITLE     Targeted Genome Editing via CRISPR in the Pathogen Cryptococcus 
            neoformans.
  JOURNAL   PLoS One. 2016 Oct 6;11(10):e0164322. doi: 
            10.1371/journal.pone.0164322. eCollection 2016.
  PUBMED    27711143
REFERENCE   2  (bases 1 to 7190)
  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: "PLoS One.";
            date: "2016-10-6"; volume: "11(10):e0164322. doi"; pages: " 
            10.1371/journal.pone.0164322. eCollection 2016"
COMMENT     Sequence Label: pSDMA66
FEATURES             Location/Qualifiers
     source          1..7190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        801..905
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(906..974,975..1766)
                     /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(1124..1143)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1937..2525
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2426..2445
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2679..2696
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2813..2834
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2849..2854,2855..2872,2873..2879)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2887..2903
                     /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     2892..2914
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2911..2927
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2911..2927
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2946..2966
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2948..2966
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     ncRNA           complement(3296..3363)
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_RNA        complement(3368..3443)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           complement(3464..3500)
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     primer_bind     complement(4407..4426)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4408..4426)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4436..4453)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4436..4452)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4445..4467)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             5575..6138
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="TLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFTLR
                     EVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRRLT
                     VEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTLCG
                     LDTALYDGTASDGEQALYMSMPCP"
ORIGIN
        1 acacgatgtt ttccatcagt aacatcgggg cacttgtaca tattctccca ggcagacaaa
       61 gatatgacaa gaccttcacg gccgacggtt gcaacatcct tctccaattt gagctgctac
      121 aatctcctgc ttcatggcat tccagtcaca tcaaggtatc catctctttc ggcatagcta
      181 atctaggaag aatgaatata ccacgtctat aactgatcag acctaaaggg cttcagtatc
      241 ggttatacac caggatcaat ggcaaataag actcacaaat tgggatgatc atcagcagat
      301 ggtgttattc aacgggcaga ccgtgagtac aaagtgttca gcgttgaatg gcaccgtata
      361 tacaaatatg ctgagcctgt tataacctta gcttcttctc ctgttgcttc ccttggcctg
      421 taaagtcaag ttccgccgtg ttctattatc ataaatgatc gacttccatc aatggaaggg
      481 taagctcgcg tccttaagca taggcggtct cagagtgcca tccagtccag cgctcaaggt
      541 tcctaggaag gggggttcgc ctttaagaat tcagggggct gtggtagctc gacgatgcca
      601 ggtgtcatgg agaggtccaa gaaattgatg ataaccattc cctagtccgc aaagtgcctc
      661 gtatgatcca aagtgccaag attgtacgat agggtggata tccaaagcaa gaagataaac
      721 gtccaattcc atgatttgaa gtctatgggc agatcttgcc gctacagggc gcgtcaggtg
      781 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
      841 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      901 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      961 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     1021 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     1081 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     1141 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     1201 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     1261 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     1321 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     1381 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     1441 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     1501 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     1561 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     1621 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     1681 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     1741 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1801 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1861 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1921 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1981 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2041 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     2101 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2161 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2221 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2281 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2341 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2401 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2461 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2521 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2581 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2641 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2701 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     2761 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2821 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2881 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2941 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctcggatcc actagtaacg
     3001 gccgccagtg tgctggaatt cgcccttggt ttatctgtat taacacggaa gagatgtaga
     3061 aactagcttc ctggtttcag agacaaggag ccatgaagat cctgaggatt caacggcgtt
     3121 gctgagggtg ggaaggatgg tattgttgtc tgtagataga ttaatgggca tataatgtca
     3181 aaaaagatga ttgacacgat gtctgtctcg aattcgcagt gtgctaaaat tgtatcactt
     3241 ctctaatatg tatatataca ccctctaagg aaaattaacc ccttaccgcc ttcacgtccc
     3301 attcgccatg ccgaagcatg ttgcccagcc ggcgccagcg aggaggctgg gaccatgccg
     3361 gccaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa
     3421 cttgctattt ctagctctaa aactaaaatc ctcttccgtc ggggacgagc ttactcgttt
     3481 cgtcctcacg gactcatcag cccgacagac atgttgggcg agtttactaa tggaaaaaga
     3541 caattagcaa agaggctgca tattaggaga cgcggtgaag attggggagg acaatagggt
     3601 ccgtgcacca aaaagtcctg acggcagctc tgttcaacgc acatccatct cttccagcta
     3661 tgcgtgtggc accagcacgg atggattgtt gcaccaaaag gacaaggcga gatggatgca
     3721 ggtcgaggcg atgcggtgct atcggccctc gttgtcgaac gccacgtcgt cctctcggcg
     3781 accgtcaatc ctggaaggaa atgaccaagc gtgcacccag catccagcat ccaacatcca
     3841 gcacccagca cctagcaccc atgatccaat ggccggcatg cagatcagcg tggggagagc
     3901 accatctagc agcccctggc aggcccagca ttgtgctcgg atgtgcgata ggcggtgggt
     3961 gcgataaggt ggattagtgg aggatggagg actgagtggc gggcaaggga agggggcggg
     4021 aagatgactc accttgggca gtgggtgggg gatgcggtat aggggatgta tgtggatgct
     4081 ggcggaggat agaagctgta agctgagctg gtgccaacgg tgacgctgtg agagtggtct
     4141 cggcgaccct gacggcgact cggcgttacc ccgtgtggca caccggacgc gcgtcgagtg
     4201 acgttgctgc tattgttgtt accatcatcc tctcctccga caaccataca ctcagtgcac
     4261 accatgccac tcgaatcctg catgcttatg tgagtcctcc ccttcccgtg cgccgctctc
     4321 cagctcacat cctcgcagca agggcgaatt ctgcagatat ccatcacact ggcggccgct
     4381 cgaggggggg cccggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     4441 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4501 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     4561 cccaacagtt gcgcagcctg aatggcgaat ggaaattgta agcgttaata ttttgttaaa
     4621 attcgcgtta aatttttgtt aaatcagctc atttttttaa ccaataggcc gaaatcggca
     4681 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtccatgg ctgcgaggat
     4741 gtgagctgga gagcggcgca cgggaagggg aggactcaca taagcatgca ggattcgagt
     4801 ggcatggtgt gcactgagtg tatggttgtc ggaggagagg atgatggtaa caacaatagc
     4861 agcaacgtca ctcgacgcgc gtccggtgtg ccacacgggg taacgccgag tcgccgtcag
     4921 ggtcgccgag accactctca cagcgtcacc gttggcacca gctcagctta cagcttctat
     4981 cctccgccag catccacata catcccctat accgcatccc ccacccactg cccaaggtga
     5041 gtcatcttcc cgcccccttc ccttgcccgc cactcagtcc tccatcctcc actaatccac
     5101 cttatcgcac ccaccgccta tcgcacatcc gagcacaatg ctgggcctgc caggggctgc
     5161 tagatggtgc tctccccacg ctgatctgca tgccggccat tggatcatgg gtgctaggtg
     5221 ctgggtgctg gatgttggat gctggatgct gggtgcacgc ttggtcattt ccttccagga
     5281 ttgacggtcg ccgagaggac gacgtggcgt tcgacaacga gggccgatag caccgcatcg
     5341 cctcgacctg catccatctc gccttgtcct tttggtgcaa caatccatcc gtgctggtgc
     5401 cacacgcata gctggaagag atggatgtgc gttgaacaga gctgccgtca ggactttttg
     5461 gtgcacggac cctattgtcc tccccaatct tcaccgcgtc tcctaatatg cagcctcttt
     5521 gctaattgtc tttttccatt agtaaactcg cccaacatgt ctatggcggc cgccactctt
     5581 gacgacacgg cttaccggta ccgcaccagt gtcccggggg acgccgaggc catcgaggca
     5641 ctggatgggt ccttcaccac cgacaccgtc ttccgcgtca ccgccaccgg ggacggcttc
     5701 accctgcggg aggtgccggt ggacccgccc ctgaccaagg tgttccccga cgacgaatcg
     5761 gacgacgaat cggacgacgg ggaggacggc gacccggact cccggacgtt cgtcgcgtac
     5821 ggggacgacg gcgacctggc gggcttcgtg gtcgtctcgt actccggctg gaaccgccgg
     5881 ctgaccgtcg aggacatcga ggtcgccccg gagcaccggg ggcacggggt cgggcgcgcg
     5941 ttgatggggc tcgcgacgga gttcgcccgc gagcggggcg ccgggcacct ctggctggag
     6001 gtcaccaacg ttaacgcacc ggctatccac gcgtaccggc ggatggggtt caccctctgc
     6061 ggcctggaca ccgccctgta cgacggcacc gcctcggacg gcgagcaggc gctctacatg
     6121 agcatgccct gcccctaaga attcgtgaag gcggtaaggg gttaattttc cttagagggt
     6181 gtatatatac atattagaga agtgatacaa ttttagcaca ctgcgaattc gagacagaca
     6241 tcgtgtcaat catctttttt gacattatat gcccattaat ctatctacag acaacaatac
     6301 catccttccc accctcagca acgccgttga atcctcagga tcttcatggc tccttgtctc
     6361 tgaaaccagg aagctagttt ctacatctct tccgtgttaa tacagataaa ccggccaatg
     6421 aagcacgctc aaaattatgc ctgtcaaaac agtcagctcc atccttttct tccctttacg
     6481 tatcatatgc tgtttttttt tttttttcga cacaagttca aagggctggc gctgcttagg
     6541 atccatcaca ccattcgtca acgtattgtg aatcaaagaa atgctcttcg atattcttta
     6601 tcagggcaag ggaacagaca gataccagca ggcttcacaa ctctgagtcg ctcggccaaa
     6661 gaggaaaaga aaagaggatg ccggcacttt ctatacagtc acaagacgcg aatgacaatc
     6721 cggcacttta tcgcatcatg tctgaatttg tgaagggact ttgagatata atcagggggt
     6781 cagacgcact ttacaactct gaatcgcttc ggagtaagga agtggaactg acagatacac
     6841 tttatagagc cttgggaacc atctggacca agaagaaggg aaaaaaaggc tacagtgagt
     6901 gagcaaccta tgctcacctt tatcctgacg ttgattgagt ctagaggcca aagatccaag
     6961 gcaaggcgaa atattccatt gtccagttca tggctgagtg accgttgttt aggttgatcg
     7021 aggcgaaaag agaagtcgat gttgatgatt gtgagaccga aatcagactt ggccgctttt
     7081 tatagttcaa aaaaggggcc cagaggtcag tgggctcgcc actcgatgca taagcaatcc
     7141 acttggcaca gactggtttg gtttaattaa cacaagtatc gtggcgcgcc
//
