LOCUS       sequence-204488-        3223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a S. pyogenes Cas9/dCas9 guide RNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3223)
  AUTHORS   Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein 
            LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford
            GE, Reddy TE, Gersbach CA
  TITLE     RNA-guided gene activation by CRISPR-Cas9-based transcription 
            factors.
  JOURNAL   Nat Methods. 2013 Jul 25. doi: 10.1038/nmeth.2600.
  PUBMED    23892895
REFERENCE   2  (bases 1 to 3223)
  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: "Nat 
            Methods. 2013 Jul 25. doi: 10.1038/nmeth.2600."
COMMENT     Sequence Label: pSPgRNA
FEATURES             Location/Qualifiers
     source          1..3223
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(377..397)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(377..395)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(416..432)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(416..432)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(429..451)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    440..456
                     /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(464..470,471..488,489..494))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    509..530
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(647..664)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(818..1406)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(898..917)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1577..2368,2369..2437))
                     /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     2200..2219
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2438..2542)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2568..3023)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2705..2726)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2917..2936
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3150..3172
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3164..3181
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3165..3181
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3191..3210
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3191..3209
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttatcg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ccgcggtgga
      361 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
      421 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
      481 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
      541 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
      601 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
      661 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
      721 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
      781 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
      841 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
      901 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
      961 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1021 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1081 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     1141 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     1201 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     1261 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     1321 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     1381 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     1441 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     1501 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     1561 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     1621 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     1681 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     1741 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     1801 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     1861 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     1921 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     1981 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     2041 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     2101 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     2161 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     2221 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     2281 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     2341 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     2401 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     2461 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2521 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     2581 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     2641 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     2701 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     2761 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     2821 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     2881 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     2941 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     3001 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
     3061 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     3121 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     3181 tgagcgcgcg taatacgact cactataggg cgaattgggt acc
//
