LOCUS       sequence-476642-        6772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of enAsCas12f-HKRA and sgRNA_v7 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6772)
  AUTHORS   Hino T, Omura SN, Nakagawa R, Togashi T, Takeda SN, Hiramoto T, 
            Tasaka S, Hirano H, Tokuyama T, Uosaki H, Ishiguro S, Kagieva M, 
            Yamano H, Ozaki Y, Motooka D, Mori H, Kirita Y, Kise Y, Itoh Y, 
            Matoba S, Aburatani H, Yachie N, Karvelis T, Siksnys V, Ohmori T, 
            Hoshino A, Nureki O
  TITLE     An AsCas12f-based compact genome-editing tool derived by deep 
            mutational scanning and structural analysis.
  JOURNAL   Cell. 2023 Sep 22:S0092-8674(23)00963-7. doi: 
            10.1016/j.cell.2023.08.031.
  PUBMED    37776859
REFERENCE   2  (bases 1 to 6772)
  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: "Cell. 2023 
            Sep 22:S0092-8674(23)00963-7. doi: 10.1016/j.cell.2023.08.031."
COMMENT     Sequence Label: pCMV enAsCas12f-HKRA-sgRNA_v7
FEATURES             Location/Qualifiers
     source          1..6772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    84..175
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     124..143
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        254..502
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     254..274
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     425..444
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    676..793
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        842..1352
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1271..1289
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1374..1973
                     /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(1374..1393)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1870..1890
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2120..2241
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2131..2150)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2185..2204
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2489..2506)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2660..3248)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2740..2759)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3419..4210,4211..4279))
                     /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     4042..4061
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4280..4384)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4411..4427
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4411..4427
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4498..4722
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4711..4728
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4751..4768)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4777..4797)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6076..6096)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      6099..6108
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6115..6134)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6116..6134)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6176..6379)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6205..6225)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6380..6759
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggcccgtaca tcgcgaagca gcgcaaaacg cctaacccta agcagattct tcatgcaatt
       61 gtcggtcaag aatcgatagt actaacatac gctctccatc aaaacaaaac gaaacaaaac
      121 aaactagcaa aataggctgt ccccagtgca agtgcaggtg ccagaacatt tctctatcga
      181 taggtaccga ttagtgaacg gatctcgacg gtatcgatca cgagactagc ctcgagcggc
      241 cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      301 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      361 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      421 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      481 cttgtggaaa ggacgaaaca ccgggattcg tcggttcagc gacgataagc cgagaagtgc
      541 caataaaact gttaagtggt ttggtaacgc tcggtaaggt ccgaaaggag aaccactgaa
      601 ccgagacccg agagagggtc ggtctccttt ttttgaattc tcgacctcga gacaaatggc
      661 agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag
      721 aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa
      781 aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc cgcggctcga
      841 gggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct
      901 ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gactcgcaca ttcttcacgt
      961 ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc
     1021 ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa
     1081 cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag
     1141 cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca
     1201 gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc
     1261 tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc
     1321 tcgttgaccg aatcaccgac ctctctcccc agggggatcc accggagctt accatgaccg
     1381 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     1441 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     1501 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     1561 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     1621 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     1681 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     1741 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     1801 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggaaacct
     1861 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     1921 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc
     1981 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg gtacctttaa
     2041 gaccaatgac ttacaaggca gctgtagatc ttagccactt tctagagtcg gggcggccgg
     2101 ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     2161 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     2221 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     2281 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat
     2341 aaggatccgt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc
     2401 gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg
     2461 acaggtgccg gcagcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2521 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2581 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2641 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2701 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2761 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2821 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2881 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2941 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3001 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3061 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3121 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3181 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3241 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3301 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3361 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3421 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3481 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3541 gtgctgcaat gataccgcgg gacccacgct caccggctcc agatttatca gcaataaacc
     3601 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3661 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3721 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3781 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3841 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3901 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3961 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4021 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4081 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4141 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4201 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4261 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4321 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4381 cgcgcacatt tccccgaaaa gtgccacctg caggaaacag ctatgaccat gattacgcca
     4441 agctctagct agaggtcgac ggtatcgagc cccagctggt tctttccgcc tcagaagcca
     4501 tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt
     4561 cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt
     4621 cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg
     4681 gggaggggca aacaacagat ggctggcaac tagaaggcac agtcgaggct gatcagcggg
     4741 tttaaactca atggtgatgg tgatgatgac cggttagact ttcctcttct tcttgggaga
     4801 accaccagac ttaatgctct cggcgatgat cttgtcgatg ttggggatgg cgatattccg
     4861 ggcggcgttg tagtcggcgt tggcctcgta gccgcaggcc cggcacttaa agatggcctg
     4921 gccgattctg ttgccggagt cgatgttgcc gcactcggag catctctggc tggtgtactg
     4981 ggggtcgatc ttgatcactt tgatgccggc ctcctcggcc ttgtagatga tcttctgctg
     5041 caggtcgtag taggtccagt tctgcaggaa tctgctgccg atgtctctga tgtttgtcag
     5101 atcctccatc tggattgtgc cgcagccctc cttgatggcc atgtccacga tgtatctgct
     5161 gtaccggtga ttggtggtgt ctctgaaatt ggcgatctta tccctcagct gctcaatggg
     5221 cttgattctc ttgtctctgc cgtggccgcc cctggcgccg ccggcgtact tgccctgtct
     5281 cagcatggag atgcgccggc tctccacctg cctcctgaag ttctcaatct cgccgccctc
     5341 cagcttgtac ctggcggggg tgtgctggaa ggccatgtaa gcggccacgg ccacgcccag
     5401 gtcaatgccc atgatcttgt tcaggtccag cactctggtc tgtggctcga atctgtagct
     5461 gatgttcagg taccacttgt tcttccggtc cttgtggata atctggctgg cgctcacctg
     5521 gtactcgccg gacaggattc tgtccatgat ggtcttgccg gcgcccctca cgatgatgat
     5581 cacggagatc tttctcttca cgttcatctc ctgcttggcg gggttgctca gcagagacag
     5641 gctggcgatg tagtcgccgt gattcatcct gttcacggag atgttctcct tgatcaggtc
     5701 caggggatgg tccctcttgt agctggggat agacatgtcg ccgcgcagga tctccttctg
     5761 gtaggccttg aacctgtctg tggctctctt gatggtctgg ctcagatttc cagagttcag
     5821 tctgtaggcc tttgtcttga tggtgtggta ggcgtagccg tgcacgttgg tgtagcccag
     5881 gatgtccttg ctcttggggt actcgccgtg gttatccttg tagtcgctgc tgaagcccat
     5941 ccactcccag gccagctgtg tggccttatt cagggcgaat ctggtttcct gctgcagctg
     6001 tctcaggatg gtgccgaact ccttccagtc caggtccaga ggcttcacga tctcgtatct
     6061 gtacaccttg atcatgactt tcctcttctt cttgggggcc atggtggcgg ctctccctat
     6121 agtgagtcgt attagcggcc gcggatctct agcggatctg acggttcact aaaccagctc
     6181 tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt
     6241 tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt
     6301 caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt
     6361 actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag
     6421 taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
     6481 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
     6541 tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg
     6601 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
     6661 catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg
     6721 taattgatta ctattaataa ctagtcaata atcaatgtca acgcgtatat ct
//
