LOCUS       sequence-336050-        3376 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of sgRNA on an optimized gRNA scaffold contains A-U pair 
            flip stabilize the CjCas9/sgRNA complex..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3376)
  AUTHORS   Zhang X, Lv S, Luo Z, Hu Y, Peng X, Lv J, Zhao S, Feng J, Huang G, 
            Wan QL, Liu J, Huang H, Luan B, Wang D, Zhao X, Lin Y, Zhou Q, Zhang
            ZN, Rong Z
  TITLE     MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional 
            activator, elicits gene expression in vivo.
  JOURNAL   Nucleic Acids Res. 2021 Mar 22. pii: 6179929. doi: 
            10.1093/nar/gkab174.
  PUBMED    33751124
REFERENCE   2  (bases 1 to 3376)
  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: "Nucleic 
            Acids Res. 2021 Mar 22. pii: 6179929. doi: 10.1093/nar/gkab174."
COMMENT     Sequence Label: pU6-cj-E sgRNA
FEATURES             Location/Qualifiers
     source          1..3376
                     /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"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..343
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     CDS             365..391
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             371..397
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     promoter        complement(415..433)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(416..433)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(451..467)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(451..467)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(464..486)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    475..491
                     /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(499..505,506..523,524..529))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    544..565
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(682..699)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(853..1441)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(933..952)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1612..2403,2404..2472))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLHKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2235..2254
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2473..2577)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2656..3110)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2792..2813)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3004..3023
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3236..3258
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3250..3267
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3251..3267
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3274..3293
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3274..3292
                     /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 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggagacggga tcccgtctcc gtttaagtcc ctgaaaaggg acttaaataa
      301 agagtttgcg ggactctgcg gggttacaat cccctaaaac cgcttttttt tcgacatcga
      361 attctatcct tatgacgtgc ctgactatgc cagcctggga ggacgcttga gtattctata
      421 gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
      481 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
      541 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
      601 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
      661 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
      721 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
      781 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
      841 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
      901 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
      961 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1021 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1081 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1141 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1201 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1261 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     1321 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     1381 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1441 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1501 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1561 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     1621 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     1681 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     1741 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     1801 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     1861 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgtgca acgttgttgc
     1921 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     1981 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     2041 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2101 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     2161 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     2221 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     2281 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     2341 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     2401 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2461 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     2521 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     2581 atttccccga aaagtgccac ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     2641 aataccgcat cagggaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     2701 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2761 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2821 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2881 tgaaccatca ctctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     2941 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3001 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3061 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca
     3121 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     3181 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     3241 tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg
     3301 attacaagga tgacgacgat gaacaaaaac tctctcagaa gaggatctgg aattcgatct
     3361 cgagtctaga ttcacc
//
