LOCUS       sequence-178968-        3395 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 promoter driven expression of Cj-SgRNA cloned with BsmbI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3395)
  AUTHORS   Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, Song DW, Lee KJ, Jung 
            MH, Kim S, Kim JH, Kim JH, Kim JS
  TITLE     In vivo genome editing with a small Cas9 orthologue derived from 
            Campylobacter jejuni.
  JOURNAL   Nat Commun. 2017 Feb 21;8:14500. doi: 10.1038/ncomms14500.
  PUBMED    28220790
REFERENCE   2  (bases 1 to 3395)
  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 
            Commun."; date: "2017-02-21"; volume: "8:14500. doi"; pages: " 
            10.1038/ncomms14500"
COMMENT     Sequence Label: pU6-Cj-sgRNA
FEATURES             Location/Qualifiers
     source          1..3395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     385..402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    519..540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(555..560,561..578,579..585)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    593..609
                     /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     598..620
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     617..633
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     617..633
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        651..669
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     651..668
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(687..713)
                     /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"
     CDS             complement(693..719)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_RNA        complement(760..832)
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     promoter        complement(854..1102)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(912..931)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1082..1102)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1186..1205)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1187..1205)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1212..1229)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1212..1228)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1221..1243)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1369..1823
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1456..1475)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1666..1687
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1902..2006
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2007..2075,2076..2867)
                     /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     complement(2225..2244)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3038..3395,1..231)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
       61 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      301 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      361 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      421 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      481 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      541 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      601 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct atttaggtga
      661 cactatagaa tactcaagcg tcctcccagg ctggcatagt caggcacgtc ataaggatag
      721 aattcgatgt cgactagtca ataatcaatg tcaaaaaaag cggttttagg ggattgtaac
      781 cccgcagagt cccgcaaact ctttatttta gtcccttttc agggactaaa acggagacgg
      841 gatcccgtct cccggtgttt cgtcctttcc acaagatata taaagccaag aaatcgaaat
      901 actttcaagt tacggtaagc atatgatagt ccattttaaa acataatttt aaaactgcaa
      961 actacccaag aaattattac tttctacgtc acgtattttg tactaatatc tttgtgttta
     1021 cagtcaaatt aattccaatt atctctctaa cagccttgta tcgtatatgc aaatatgaag
     1081 gaatcatggg aaataggccc tcggtgaatc tagactcgag atcgaattcc agatcctctt
     1141 ctgagagagt ttttgttcat cgtcgtcatc cttgtaatca attcgcccta tagtgagtcg
     1201 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     1261 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     1321 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta
     1381 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1441 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1501 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1561 acctcgaccc caaaaaactt gattagagtg atggttcacg tagtgggcca tcgccctgat
     1621 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1681 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1741 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1801 acaaaatatt aacgcttaca attccctgat gcggtatttt ctccttacgc atctgtgcgg
     1861 tatttcacac cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1921 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1981 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2041 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2101 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2161 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2221 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     2281 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2341 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2401 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2461 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2521 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cacaaactat
     2581 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2641 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2701 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     2761 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2821 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2881 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2941 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3001 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3061 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3121 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3181 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3241 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3301 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3361 ggggttcgtg cacacagccc agcttggagc gaacg
//
