LOCUS       sequence-405077-        6914 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing a CMV-driven 
            NLS-AcCas12n-PT1-NLS(human-codonoptimized)cassette and a U6-driven 
            sgRNA_V6 cassette(containing BsaI sites for spacer insertion). 
            Carbenicillin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6914)
  AUTHORS   Chen W, Ma J, Wu Z, Wang Z, Zhang H, Fu W, Pan D, Shi J, Ji Q
  TITLE     Cas12n nucleases, early evolutionary intermediates of type V CRISPR,
            comprise a distinct family of miniature genome editors.
  JOURNAL   Mol Cell. 2023 Jun 27:S1097-2765(23)00463-X. doi: 
            10.1016/j.molcel.2023.06.014.
  PUBMED    37402371
REFERENCE   2  (bases 1 to 6914)
  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: "Mol Cell. 
            2023 Jun 27:S1097-2765(23)00463-X. doi: 
            10.1016/j.molcel.2023.06.014."
COMMENT     Sequence Label: pAcCas12nHS_RV6_PT1_empty
FEATURES             Location/Qualifiers
     source          1..6914
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    567..684
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        733..1243
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1162..1180
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1265..1864
                     /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(1265..1284)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1761..1781
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2011..2132
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2022..2041)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2076..2095
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2380..2397)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2551..3139)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2631..2650)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3310..4101,4102..4170))
                     /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     3933..3952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4171..4275)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4302..4318
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4302..4318
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4389..4613
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4602..4619
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4642..4659)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4668..4688)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      5729..5738
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(6036..6056)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      6059..6068
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6075..6094)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6076..6094)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6136..6339)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6165..6185)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6340..6719
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    6816..6907
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6856..6875
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc gcagtgctga tcgatcgaaa cgtcgcctgc gataggcggg
      361 agacgctaaa cgcccgtgga gcatccataa gaccaaccac ctctcggggc ggtaggcacg
      421 acgcatcgaa gcgggaaggc tccggcgctc ggcctgagtc acctcagcag agtgatctgc
      481 tgacgctcga aagagcgatc gcgagaccat tggtctcatt tttttgaatt ctcgacctcg
      541 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
      601 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
      661 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg
      721 ccgcggctcg agggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg
      781 acgcggctgc tctgggcgtg gttccgggaa acgcagcggc gccgaccctg ggactcgcac
      841 attcttcacg tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc
      901 ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg
      961 gacgtgacaa acggaagccg cacgtctcac tagtaccctc gcagacggac agcgccaggg
     1021 agcaatggca gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcagggcg
     1081 cgccgagagc agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg
     1141 ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag cctccggagc gcacgtcggc
     1201 agtcggctcc ctcgttgacc gaatcaccga cctctctccc cagggggatc caccggagct
     1261 taccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     1321 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     1381 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     1441 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     1501 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     1561 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     1621 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     1681 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     1741 cctggaaacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     1801 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     1861 ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc
     1921 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttctagagtc
     1981 ggggcggccg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
     2041 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     2101 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     2161 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     2221 gtaaaatcga taaggatccg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     2281 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     2341 caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg
     2401 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2461 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     2521 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2581 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2641 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2701 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     2761 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2821 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2881 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2941 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3001 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3061 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3121 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3181 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3241 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3301 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3361 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3421 atctggcccc agtgctgcaa tgataccgcg ggacccacgc tcaccggctc cagatttatc
     3481 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3541 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3601 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3661 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3721 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3781 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3841 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3901 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3961 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4021 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4081 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4141 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4201 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4261 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gcaggaaaca gctatgacca
     4321 tgattacgcc aagctctagc tagaggtcga cggtatcgag ccccagctgg ttctttccgc
     4381 ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc
     4441 ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg
     4501 cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa
     4561 ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc
     4621 tgatcagcgg gtttaaactc aatggtgatg gtgatgatga ccggttagac tttcctcttc
     4681 ttcttgggag aaccaccaga cctatcaatc aacacagcgt gcctctcgat gttcctagcg
     4741 gcgttcatat ctctatccat gaccaggccg cagtcggtgc acctaaacac cctatcggag
     4801 agcttcaagt gggggttctg ccagccgcac ccgctgcagg tcttgctgga ggggaaccac
     4861 ctgtcggcca cggccagctc gctcccgtac caggtggcct tgtagttcac ctgcctccgc
     4921 agctcggcag gggcgctgtc caggatgacc cggttcagcc cggccttctg ccggacgttc
     4981 ttccctgggg cggccactgt ccccctggcg gacctggtca tgccggcgac gttcagatcc
     5041 tccagggcca ccacggcgaa ccctgtggtc agccgcttgg tgatctggtg cagggcggcg
     5101 tgccgccgga cggccacctc gtgatgggcc ctccccaccc gccgcttggc cttctctctc
     5161 ctggcgctgc ccttctgtgt cctggccagg gcccgctggg ccttgagcag ccgcctctca
     5221 gcctgcctca ggtgctgggg gtttctcacc agggcgctgc ttgggtcgtc gacgaccagg
     5281 ggcttggaca gggcggccag caccttcacc cccaggtcca ctcccacggc gcccctctcc
     5341 cgctgccgcc tgctgggccg atcgggcacc tgcactgtca ccttgcagag cacggaggcg
     5401 taccacctgt gtccgcccct actcacagtc acggactgca ccacggcgtc gcctctgtca
     5461 atgagcctgg cgagccgctt gccgctgtcg tgcagcctca cctcgccaat cctaggcagc
     5521 ctcagccttc tgtagccagc cagcctgatg ctgggcttct tcacatcgtg gtgcaatcta
     5581 aagctatccc gagcccggcc cttcttctta aaccgggggt agcccaccct cctccccact
     5641 ctcttgccgc tcaggctgtc gagccagttc ttccatgcct ggtcggcgtc aataaaggcg
     5701 ctctggaagg cgtaggtgtt cacctcgtgc caccatgggc aggccccatc caggcccttc
     5761 cgggaatccc ccttcacggc gttcagggcc ttctggatgg ctggcttcat aggcacgggg
     5821 acccggacct gccgccgggc ctgctcctcg ggcacgccat cgcccacgag cttggccacc
     5881 tcggcccgcc accgctcgtg ggcccccacc ttggcggcga gggcgtggtt gaaggcccac
     5941 ctggcggccc cggcgtgccg gctcagggcg gccacctgga cggaagtagg atcgagggcg
     6001 aaccgaaagg ccttcaggat ctctgtctgc accatgacct tcctcttctt cttaggggcc
     6061 atggtggcgg ctctccctat agtgagtcgt attagcggcc gcggatctct agcggatctg
     6121 acggttcact aaaccagctc tgcttatata gacctcccac cgtacacgcc taccgcccat
     6181 ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca
     6241 aaacaaactc ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg
     6301 ctatccacgc ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa
     6361 tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca
     6421 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
     6481 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     6541 ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     6601 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     6661 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca
     6721 acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt
     6781 cttcatgcaa ttgtcggtca agaatcgata gtactaacat acgctctcca tcaaaacaaa
     6841 acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg tgccagaaca
     6901 tttctctatc gata
//
