LOCUS       sequence-390948-        7069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmid expresses human codon-optimized Cas12j-8, crRNA 
            expression elements and puromycin resistence gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7069)
  AUTHORS   Wang Y, Qi T, Liu J, Yang Y, Wang Z, Wang Y, Wang T, Li M, Li M, Lu 
            D, Chang ACY, Yang L, Gao S, Wang Y, Lan F
  TITLE     A highly specific CRISPR-Cas12j nuclease enables allele-specific 
            genome editing.
  JOURNAL   Sci Adv. 2023 Feb 10;9(6):eabo6405. doi: 10.1126/sciadv.abo6405. 
            Epub 2023 Feb 10.
  PUBMED    36763662
REFERENCE   2  (bases 1 to 7069)
  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: "Sci Adv."; 
            date: "2023-02-10"; volume: "9(6):eabo6405. doi"; pages: " 
            10.1126/sciadv.abo6405. Epub 2023 Feb 10"
COMMENT     Sequence Label: Cas12j-8-puro-crRNA
FEATURES             Location/Qualifiers
     source          1..7069
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      756..765
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             768..788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2961..3008
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3030..3086
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3087..3683
                     /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(3087..3106)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3583..3603
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3684..3764
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3792..3809)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3798..4005
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4012..4260
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4012..4032
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4183..4202
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   4332..4472
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4332..4461
                     /label=AAV2 ITR
     rep_origin      4547..5002
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4634..4653)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4844..4865
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5019..5038)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5138..5160
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5198..5216)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5284..5388
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5389..5457,5458..6249)
                     /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(5607..5626)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6420..7008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6909..6928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc
      781 ggaaggtcgg tatccacgga gtcccagcag ccatcaagcc aaccgtctcc cagttcctga
      841 cacctggctt taagctgatc agaaaccaca gcaggacagc cggcctgaag ctgaagaatg
      901 agggagaaga agcttgtaaa aaattcgtgc gggagaacga gatccctaaa gacgagtgcc
      961 ccaacttcca gggcggccct gccatcgcta acatcatcgc caagagcaga gaatttaccg
     1021 agtgggagat ctaccagtct agcctggcta ttcaggaggt gatcttcacc ctgcctaaag
     1081 ataagctgcc tgaacctatc ctgaaggaag aatggagagc ccaatggctg tctgagcacg
     1141 gcctggacac cgttccttac aaggaggccg ccggactcaa cctgatcatc aagaacgccg
     1201 tgaataccta caagggcgtg caggtgaagg tggacaacaa gaacaagaac aacctggcca
     1261 agatcaatag aaagaatgag atcgccaagc tgaacggaga gcaggagatc agcttcgagg
     1321 aaatcaaggc ctttgacgac aagggatacc tgctgcagaa gcccagccct aacaagtcca
     1381 tctattgcta ccaatctgtg tccccaaagc ccttcatcac cagcaaatac cacaacgtga
     1441 acctgccaga ggagtacatc ggctactaca gaaagtctaa tgaacccatc gtgtctccct
     1501 accagttcga tagactgcgg atccccatcg gcgagcctgg atatgtgcct aagtggcagt
     1561 acaccttcct gtctaagaag gaaaacaaac ggagaaagct ttctaaaagg atcaagaatg
     1621 taagccccat cctgggcatc atttgtatca aaaaggactg gtgcgtgttc gacatgcggg
     1681 gactgctgag aaccaaccat tggaagaagt accacaagcc tacagatagc atcaatgatc
     1741 tgttcgacta ttttacaggc gaccccgtga tcgacacaaa ggccaatgtg gtcagattcc
     1801 ggtacaagat ggaaaatggc atcgtgaatt acaaacccgt gcgcgaaaaa aagggcaaag
     1861 agctgctgga aaacatttgc gatcaaaacg gtagctgcaa gctggctaca gtcgatgtgg
     1921 gccagaacaa ccccgtggcc atcggcctct tcgagctgaa gaaggtgaac ggcgagctga
     1981 ccaagacact gatcagcaga cacccaaccc ctatcgactt ctgcaacaag atcaccgcct
     2041 accgggaaag atacgacaag ctggaaagct ctattaagct ggacgccatc aagcagctga
     2101 ccagcgagca gaaaatcgag gtggacaact acaacaacaa cttcacccct cagaacacca
     2161 agcagatcgt gtgcagcaag ctgaacatca accccaacga cctgccttgg gacaagatga
     2221 tcagcggcac acacttcatc tctgaaaagg cacaggtgag caacaagtct gagatctact
     2281 tcacctccac cgacaagggc aagacaaagg acgtgatgaa gagcgactac aagtggtttc
     2341 aagactacaa gcctaagctg tctaaggaag tgcgagacgc cctgtcagat atcgagtggc
     2401 ggctgagaag agagagcctg gaattcaaca aactgagcaa aagcagagag caggacgcca
     2461 gacagctggc caactggatc agcagcatgt gcgatgtgat cggcatcgag aacctggtga
     2521 aaaagaacaa cttcttcggc ggcagcggca aacgggaacc tggctgggat aatttctaca
     2581 agcctaagaa ggaaaaccgg tggtggatca acgccatcca caaggctctg actgaactgt
     2641 cccagaacaa gggcaagaga gtgatcttac tgcctgctat gcggaccagc atcacctgtc
     2701 ctaagtgtaa atactgtgat agcaagaaca gaaacggcga gaagttcaac tgcctgaagt
     2761 gcggcatcga gctgaacgcc gacattgatg ttgctacaga gaatctggcc accgtggcca
     2821 ttaccgcgca gtccatgcct aagcccacct gcgagagaag cggcgatgcc aagaaaccgg
     2881 tccgcgccag aaaggccaaa gcccctgagt ttcacgacaa gctcgctcct agctacacag
     2941 tcgtgctgag agaggccgtg aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa
     3001 agaaaaaggg atccgctagc ggcagcggcg ccaccaactt cagcctgctg aagcaggccg
     3061 gcgacgtgga ggagaacccc ggccccatga ccgagtacaa gcccacggtg cgcctcgcca
     3121 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     3181 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     3241 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     3301 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     3361 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     3421 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc
     3481 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     3541 agcgcgccgg ggtgcccgcc ttcctggaaa cctccgcgcc ccgcaacctc cccttctacg
     3601 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     3661 gcatgacccg caagcccggt gcctacccat acgatgttcc agattacgct tacccatacg
     3721 atgttccaga ttacgcttac ccatacgatg ttccagatta cgcttaagaa ttcctagagc
     3781 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3841 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3901 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3961 cagcaagggg gaggattggg aagagaatag caggcatgct ggggaggtac cgagggccta
     4021 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
     4081 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
     4141 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
     4201 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
     4261 gctttcaaga ctaatagatt gctccttacg aggagaccag tcttctcaga agacgttttt
     4321 tttgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     4381 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     4441 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     4501 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     4561 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4621 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4681 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4741 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     4801 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4861 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     4921 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4981 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     5041 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5101 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5161 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     5221 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     5281 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5341 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5401 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5461 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5521 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5581 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5641 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5701 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5761 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5821 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5881 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5941 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     6001 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     6061 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt
     6121 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6181 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6241 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6301 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6361 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6421 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6481 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6541 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6601 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6661 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6721 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6781 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6841 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6901 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6961 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7021 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
