LOCUS       sequence-385765-        4673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cj-SgRNA scaffold cloned with BbsI and puromycin 
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4673)
  AUTHORS   Gao S, Wang Y, Qi T, Wei J, Hu Z, Liu J, Sun S, Liu H, Wang Y
  TITLE     Genome editing with natural and engineered CjCas9 orthologs.
  JOURNAL   Mol Ther. 2023 Apr 5;31(4):1177-1187. doi: 
            10.1016/j.ymthe.2023.01.029. Epub 2023 Feb 1.
  PUBMED    36733251
REFERENCE   2  (bases 1 to 4673)
  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"; doi: 
            "10.1016/j.ymthe.2023.01.029"; journalName: "Mol Ther"; date: 
            "2023-04-5- 5"; volume: "31"; issue: "4"; pages: "1177-1187"
COMMENT     Sequence Label: pSK-mU6-Cj-sgRNA-SV40-puro
FEATURES             Location/Qualifiers
     source          1..4673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(94..113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     304..325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     589..611
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     603..620
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     604..620
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        complement(690..762)
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     promoter        complement(782..1094)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(843..863)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        1135..1464
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1315..1450
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1474..2073
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1474..1493)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1970..1990
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2286..2420
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2323..2342)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2377..2396
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2435..2451)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2435..2451)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2488..2508)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2488..2506)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2527..2543)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2527..2543)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2540..2562)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2551..2567
                     /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(2575..2581,2582..2599,2600..2605))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2620..2641
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2758..2775)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2929..3517)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3009..3028)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3688..4479,4480..4548))
                     /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     4311..4330
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4549..4653)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
       61 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      361 atttataagg gattttgccg atttcggcct attggttaaa aaaatgagct gatttaacaa
      421 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 ccgggccccc cctcgagact agtaaaaaag cggttttagg ggattgtaac cccgcagagt
      721 cccgcaaact ctttatttta gtcccttttc agggactaaa acgtcttctg agaagactgc
      781 aaacaaggct tttctccaag ggatatttat agtctcaaaa cacacaatta ctttacagtt
      841 agggtgagtt tccttttgtg ctgtttttta aaataataat ttagtatttg tatctcttat
      901 agaaatccaa gcctatcatg taaaatgtag ctagtattaa aaagaacaga ttatctgtct
      961 tttatcgcac attaagcctc tatagttact aggaaatatt atatgcaaat taaccggggc
     1021 aggggagtag ccgagcttct cccacaagtc tgtgcgaggg ggccggcgcg ggcctagaga
     1081 tggcgcgtcg gatctctaga gcgggcccgg tactaaccaa actggatatc gatagtgtgt
     1141 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     1201 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     1261 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     1321 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     1381 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     1441 tttggaggcc taggcttttg caaaaagctt accatgaccg agtacaagcc cacggtgcgc
     1501 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     1561 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     1621 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     1681 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     1741 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     1801 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     1861 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     1921 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     1981 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     2041 acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg
     2101 accgaaagga gcgcacgacc ccatgcatcg aaatgaccga ccaagcgacg cccaacctgc
     2161 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     2221 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     2281 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     2341 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     2401 tatcttatca tgtctggatc cctgcagccc gggggatcca ctagttctag agcggccgcc
     2461 accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     2521 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2581 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2641 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2701 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2761 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2821 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2881 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2941 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3001 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3061 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3121 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3181 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3241 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3301 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3361 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3421 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3481 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3541 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3601 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3661 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3721 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3781 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     3841 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3901 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3961 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4021 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4081 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4141 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4201 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4261 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4321 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4381 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4441 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4501 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4561 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4621 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgc
//
