LOCUS       sequence-341510-        6845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AsCas12f1-based Human cell genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6845)
  AUTHORS   Wu Z, Zhang Y, Yu H, Pan D, Wang Y, Wang Y, Li F, Liu C, Nan H, Chen
            W, Ji Q
  TITLE     Programmed genome editing by a miniature CRISPR-Cas12f nuclease.
  JOURNAL   Nat Chem Biol. 2021 Sep 2. pii: 10.1038/s41589-021-00868-6. doi: 
            10.1038/s41589-021-00868-6.
  PUBMED    34475565
REFERENCE   2  (bases 1 to 6845)
  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 Chem 
            Biol. 2021 Sep 2. pii: 10.1038/s41589-021-00868-6. doi: 
            10.1038/s41589-021-00868-6."
COMMENT     Sequence Label: pCMV-AsCas12f1
FEATURES             Location/Qualifiers
     source          1..6845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     639..658
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        639..657
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      665..674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             677..697
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1976..1996
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2005..2022
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2045..2062)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2051..2275
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2346..2362)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2346..2362)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2389..2493
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2494..2562,2563..3354)
                     /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(2712..2731)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3525..4113
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4014..4033
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4267..4284
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    4532..4653
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4569..4588)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4623..4642
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(4800..5399)
                     /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(4883..4903)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5380..5399
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(5421..5931)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5484..5502)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    5980..6097
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(6344..6592)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(6402..6421)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(6572..6592)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    6671..6762
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(6703..6722)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
      601 ttagtgaacc gtcagatccg ctagagatcc gcggccgcta atacgactca ctatagggag
      661 agccgccacc atggccccca agaagaagag gaaagtcatg atcaaggtgt acagatacga
      721 gatcgtgaag cctctggacc tggactggaa ggagttcggc accatcctga gacagctgca
      781 gcaggaaacc agattcgccc tgaataaggc cacacagctg gcctgggagt ggatgggctt
      841 cagcagcgac tacaaggata accacggcga gtaccccaag agcaaggaca tcctgggcta
      901 caccaacgtg cacggctacg cctaccacac catcaagaca aaggcctaca gactgaactc
      961 tggaaatctg agccagacca tcaagagagc cacagacagg ttcaaggcct accagaagga
     1021 gatcctgcgc ggcgacatgt ctatccccag ctacaagagg gacatccccc tggacctgat
     1081 caaggagaac atctccgtga acaggatgaa tcacggcgac tacatcgcca gcctgtctct
     1141 gctgagcaac cccgccaagc aggagatgaa cgtgaagaga aagatctccg tgatcatcat
     1201 cgtgaggggc gccggcaaga ccatcatgga cagaatcctg tccggcgagt accaggtgag
     1261 cgccagccag attatccacg acgaccggaa gaacaagtgg tacctgaaca tcagctacga
     1321 cttcgagcca cagaccagag tgctggacct gaacaagatc atgggcattg acctgggcgt
     1381 ggccgtggcc gtgtacatgg ccttccagca cacccccgcc aggtacaagc tggagggcgg
     1441 cgagattgag aacttcagga ggcaggtgga gagccggcgc atctccatgc tgagacaggg
     1501 caagtacgcc ggcggcgcca ggggcggcca cggcagagac aagagaatca agcccattga
     1561 gcagctgagg gataagatcg ccaatttcag agacaccacc aatcaccggt acagcagata
     1621 catcgtggac atggccatca aggagggctg cggcacaatc cagatggagg atctgacaaa
     1681 catcagagac atcggcagca gattcctgca gaactggacc tactacgacc tgcagcagaa
     1741 gatcatctac aaggccgagg aggccggcat caaagtgatc aagatcgacc cccagtacac
     1801 cagccagaga tgctccgagt gcggcaacat cgactccggc aacagaatcg gccaggccat
     1861 ctttaagtgc cgggcctgcg gctacgaggc caacgccgac tacaacgccg cccggaatat
     1921 cgccatcccc aacatcgaca agatcatcgc cgagagcatt aagtctggtg gttctcccaa
     1981 gaagaagagg aaagtctaac cggtcatcat caccatcacc attgagttta aacccgctga
     2041 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2101 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2161 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2221 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2281 gaggcggaaa gaaccagctg gggctcgata ccgtcgacct ctagctagag cttggcgtaa
     2341 tcatggtcat agctgtttcc tgcaggtggc acttttcggg gaaatgtgcg cggaacccct
     2401 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2461 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2521 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2581 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2641 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2701 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2761 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2821 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2881 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2941 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3001 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     3061 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3121 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3181 gctgataaat ctggagccgg tgagcgtggg tcccgcggta tcattgcagc actggggcca
     3241 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3301 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3361 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3421 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3481 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3541 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3601 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3661 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3721 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3781 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3841 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3901 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3961 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4021 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4081 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4141 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4201 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4261 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgct gccggcacct gtcctacgag
     4321 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     4381 gaaggagctg actgggttga aggctctcaa gggcatcggt cgacggatcc ttatcgattt
     4441 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     4501 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     4561 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4621 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgctcgaagc ggccggccgc
     4681 cccgactcta gaaagtggct aagatctaca gctgccttgt aagtcattgg tcttaaaggt
     4741 accgatgcat ggggtcgtgc gctcctttcg gtcgggcgct gcgggtcgtg gggcgggcgt
     4801 caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac ctcgacgtcg
     4861 gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc ggaggtttcc
     4921 aggaaggcgg gcaccccggc gcgctcggcc gcctccactc cggggagcac gacggcgctg
     4981 cccagaccct tgccctggtg gtcgggcgag acgccgacgg tggccaggaa ccacgcgggc
     5041 tccttgggcc ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc cagccgggaa
     5101 ccgctcaact cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc ttcgacgctc
     5161 tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg cgacccacac cttgccgatg
     5221 tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc gatgtggcgg
     5281 tccggatcga cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc gagggtgcgt
     5341 acggccctgg ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta ctcggtcatg
     5401 gtaagctccg gtggatcccc ctggggagag aggtcggtga ttcggtcaac gagggagccg
     5461 actgccgacg tgcgctccgg aggcttgcag aatgcggaac accgcgcggg caggaacagg
     5521 gcccacacta ccgccccaca ccccgcctcc cgcaccgccc cttcccggcc gctgctctcg
     5581 gcgcgccctg ctgagcagcc gctattggcc acagcccatc gcggtcggcg cgctgccatt
     5641 gctccctggc gctgtccgtc tgcgagggta ctagtgagac gtgcggcttc cgtttgtcac
     5701 gtccggcacg ccgcgaaccg caaggaacct tcccgactta ggggcggagc aggaagcgtc
     5761 gccggggggc ccacaagggt agcggcgaag atccgggtga cgctgcgaac ggacgtgaag
     5821 aatgtgcgag tcccagggtc ggcgccgctg cgtttcccgg aaccacgccc agagcagccg
     5881 cgtccctgcg caaacccagg gctgccttgg aaaaggcgca accccaaccc cctcgagccg
     5941 cggccaaagt ggatctctgc tgtccctgta ataaacccga aaattttgaa tttttgtaat
     6001 ttgtttttgt aattctttag tttgtatgtc tgttgctatt atgtctacta ttctttcccc
     6061 tgcactgtac cccccaatcc ccccttttct tttaaaattg tggatgaata ctgccatttg
     6121 tctcgaggtc gagaattcaa aaaaaggaga ccgaccctct ctcgggtctc ggttcacact
     6181 ccacaagcta gctcgcaaac ccttgtttat gagctatatg tgaagcgtcc gtgcggtctt
     6241 tgtgcacgga gtttcagcct tttggctacc ttaccgagcg ttaccaaacc acttaacagt
     6301 tttattggca cttctcggct tatcgtcgct gaaccgacga atcggtgttt cgtcctttcc
     6361 acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc atatgatagt
     6421 ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac tttctacgtc
     6481 acgtattttg tactaatatc tttgtgttta cagtcaaatt aattccaatt atctctctaa
     6541 cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc tcggtgaagg
     6601 gggcggccgc tcgaggctag tctcgtgatc gataccgtcg agatccgttc actaatcggt
     6661 acctatcgat agagaaatgt tctggcacct gcacttgcac tggggacagc ctattttgct
     6721 agtttgtttt gtttcgtttt gttttgatgg agagcgtatg ttagtactat cgattcttga
     6781 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     6841 gggcc
//
