LOCUS       sequence-341489-        6922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AsCas12f1 heterologous expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6922)
  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 6922)
  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
FEATURES             Location/Qualifiers
     source          1..6922
                     /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             1882..1899
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1952..1970)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1966..2013
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2050..2505
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2137..2156)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2347..2368
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2597..3412)
                     /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(2597..3412)
                     /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     3320..3339
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3404..3423
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3534..4122
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4023..4042
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4276..4293
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4308..4447
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4373..4392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4533..4555
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4549..4740)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5549..6631)
                     /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     6593..6612
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6632..6709)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6915..6922,1..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 ggacccatga tcaaagttta ccgttatgaa atcgttaaac
      601 cgctggacct ggattggaaa gaattcggca ccatcctgcg tcagctgcag caggaaaccc
      661 gcttcgcgct gaacaaagca acccagctgg cgtgggaatg gatgggtttc agctctgatt
      721 acaaagacaa ccatggcgaa tacccgaaaa gcaaagacat cctgggctac accaacgttc
      781 acggttacgc ataccacacc atcaaaacca aagcttatcg tctgaacagc ggcaacctga
      841 gccagaccat caaacgtgcg accgatcgct tcaaagcgta ccagaaagaa atcctgcgtg
      901 gtgacatgtc catcccgtct tacaaacgtg atatcccgct ggatctgatc aaagaaaaca
      961 tcagcgttaa ccgtatgaac cacggtgatt atatcgcttc cctgtccctg ctgtctaacc
     1021 cggcgaaaca ggaaatgaac gttaaacgta aaatctccgt gattatcatc gttcgtggcg
     1081 cgggtaaaac catcatggat cgcatcctga gcggcgaata ccaggtgagc gcgagccaga
     1141 tcatccacga cgaccgtaaa aacaaatggt acctgaacat ctcttatgat ttcgaaccgc
     1201 agacccgtgt tctggatctg aacaaaatca tgggcatcga tctgggtgtt gcggtggcgg
     1261 tgtacatggc gttccagcac accccggcgc gctacaaact ggaaggcggt gaaatcgaaa
     1321 acttccgtcg tcaggtggag tctcgtcgca tctccatgct gcgccagggt aaatacgcgg
     1381 gtggtgctcg tggcggtcac ggccgtgata aacgtatcaa accgatcgaa cagctgcgtg
     1441 acaaaatcgc taacttccgc gataccacta accaccgtta ctctcgttac atcgttgata
     1501 tggctatcaa agaaggttgc ggtaccattc agatggaaga tctgaccaac atccgtgata
     1561 tcggctctcg tttcctgcag aactggacct actacgatct gcagcagaaa atcatctaca
     1621 aagcggaaga agcgggcatc aaagttatca aaattgatcc gcagtacacc agccagcgtt
     1681 gcagcgaatg cggcaacatc gattccggta accgtatcgg tcaggcgatc ttcaaatgcc
     1741 gtgcttgcgg ctacgaagcg aacgcggact acaacgcagc tcgtaacatc gcgatcccga
     1801 acatcgataa aatcatcgcg gaaagcatca aataagctta agaattcgag ctccgtcgac
     1861 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     1921 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1981 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     2041 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2101 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2161 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2221 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2281 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2341 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2401 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2461 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     2521 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2581 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2641 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2701 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     2761 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2821 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2881 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2941 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     3001 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     3061 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     3121 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3181 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3241 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3301 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3361 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3421 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     3481 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3541 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3601 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3661 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3721 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3781 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3841 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3901 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3961 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4021 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4081 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4141 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4201 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4261 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4321 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4381 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4441 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4501 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4561 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4621 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4681 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4741 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4801 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4861 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4921 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4981 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5041 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5101 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5161 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5221 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5281 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5341 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5401 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5461 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5521 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5581 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5641 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5701 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     5761 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     5821 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     5881 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     5941 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6001 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6061 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6121 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6181 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6241 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6301 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6361 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6421 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6481 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6541 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6601 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6661 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     6721 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     6781 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     6841 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     6901 cgatcttccc catcggtgat gt
//
