LOCUS       sequence-341508-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NLS-AsCas12f1-NLS heterologous expression plasmid for RNP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6973)
  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 6973)
  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: pET28a-SUMO-AsCas12f-2NLS
FEATURES             Location/Qualifiers
     source          1..6973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     96..115
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        96..114
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    115..139
                     /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)."
     RBS             154..176
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             195..212
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             222..239
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             240..533
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREKIGG"
     CDS             543..566
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             564..584
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1863..1883
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1933..1950
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2003..2021)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2017..2064
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2101..2556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2188..2207)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2398..2419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2648..3463)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2648..3463)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3371..3390
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3455..3474
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3585..4173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4074..4093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4327..4344
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4359..4498
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4424..4443)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4584..4606
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4600..4791)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5600..6682)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6644..6663
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6683..6760)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6966..6973,1..12)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
       61 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
      121 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
      181 ccatgggcag cagccatcat catcatcatc acagcagcgg cctggtgccg cgcggcagca
      241 tgtcggactc agaagtcaat caagaagcta agccagaggt caagccagaa gtcaagcctg
      301 agactcacat caatttaaag gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa
      361 agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggg aaggaaatgg
      421 actccttaag attcttgtac gacggtatta gaattcaagc tgatcagacc cctgaagatt
      481 tggacatgga ggataacgat attattgagg ctcacagaga aaagattggt ggtcatagta
      541 gtcttgaagt cctctttcag ggacccaaga agaagaggaa agtcatgatc aaagtttacc
      601 gttatgaaat cgttaaaccg ctggacctgg attggaaaga attcggcacc atcctgcgtc
      661 agctgcagca ggaaacccgc ttcgcgctga acaaagcaac ccagctggcg tgggaatgga
      721 tgggtttcag ctctgattac aaagacaacc atggcgaata cccgaaaagc aaagacatcc
      781 tgggctacac caacgttcac ggttacgcat accacaccat caaaaccaaa gcttatcgtc
      841 tgaacagcgg caacctgagc cagaccatca aacgtgcgac cgatcgcttc aaagcgtacc
      901 agaaagaaat cctgcgtggt gacatgtcca tcccgtctta caaacgtgat atcccgctgg
      961 atctgatcaa agaaaacatc agcgttaacc gtatgaacca cggtgattat atcgcttccc
     1021 tgtccctgct gtctaacccg gcgaaacagg aaatgaacgt taaacgtaaa atctccgtga
     1081 ttatcatcgt tcgtggcgcg ggtaaaacca tcatggatcg catcctgagc ggcgaatacc
     1141 aggtgagcgc gagccagatc atccacgacg accgtaaaaa caaatggtac ctgaacatct
     1201 cttatgattt cgaaccgcag acccgtgttc tggatctgaa caaaatcatg ggcatcgatc
     1261 tgggtgttgc ggtggcggtg tacatggcgt tccagcacac cccggcgcgc tacaaactgg
     1321 aaggcggtga aatcgaaaac ttccgtcgtc aggtggagtc tcgtcgcatc tccatgctgc
     1381 gccagggtaa atacgcgggt ggtgctcgtg gcggtcacgg ccgtgataaa cgtatcaaac
     1441 cgatcgaaca gctgcgtgac aaaatcgcta acttccgcga taccactaac caccgttact
     1501 ctcgttacat cgttgatatg gctatcaaag aaggttgcgg taccattcag atggaagatc
     1561 tgaccaacat ccgtgatatc ggctctcgtt tcctgcagaa ctggacctac tacgatctgc
     1621 agcagaaaat catctacaaa gcggaagaag cgggcatcaa agttatcaaa attgatccgc
     1681 agtacaccag ccagcgttgc agcgaatgcg gcaacatcga ttccggtaac cgtatcggtc
     1741 aggcgatctt caaatgccgt gcttgcggct acgaagcgaa cgcggactac aacgcagctc
     1801 gtaacatcgc gatcccgaac atcgataaaa tcatcgcgga aagcatcaaa tctggtggtt
     1861 ctcccaagaa gaagaggaaa gtctaagctt aagaattcga gctccgtcga caagcttgcg
     1921 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     1981 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     2041 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     2101 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2161 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2221 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2281 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2341 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2401 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2461 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2521 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     2581 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     2641 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     2701 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     2761 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     2821 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     2881 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     2941 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     3001 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     3061 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     3121 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     3181 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     3241 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     3301 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     3361 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     3421 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     3481 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     3541 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3601 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3661 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3721 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3781 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3841 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3901 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3961 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4021 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4081 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4141 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4201 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4261 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4321 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     4381 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     4441 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4501 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4561 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4621 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4681 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     4741 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     4801 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4861 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4921 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4981 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     5041 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     5101 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     5161 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     5221 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     5281 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     5341 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     5401 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     5461 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     5521 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     5581 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5641 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5701 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5761 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5821 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5881 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5941 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     6001 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     6061 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     6121 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     6181 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     6241 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     6301 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     6361 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     6421 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     6481 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     6541 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     6601 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     6661 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     6721 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     6781 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     6841 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6901 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6961 ccatcggtga tgt
//
