LOCUS       sequence-394112-        7067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector encoding a human codon-optimized enOsCas12f1 driven by CBh 
            promoter, guide RNAs compatible with enOsCas12f1 driven by hU6, and 
            mCherry driven by CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7067)
  AUTHORS   Kong X, Zhang H, Li G, Wang Z, Kong X, Wang L, Xue M, Zhang W, Wang 
            Y, Lin J, Zhou J, Shen X, Wei Y, Zhong N, Bai W, Yuan Y, Shi L, Zhou
            Y, Yang H
  TITLE     Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and
            expanded target range for genome editing.
  JOURNAL   Nat Commun. 2023 Apr 11;14(1):2046. doi: 10.1038/s41467-023-37829-7.
  PUBMED    37041195
REFERENCE   2  (bases 1 to 7067)
  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 
            Commun."; date: "2023-04-11"; volume: "14(1):2046. doi"; pages: " 
            10.1038/s41467-023-37829-7"
COMMENT     Sequence Label: pCBh_3x Flag-NLS-enOsCas12f1-NLS_pA_phU6_gRNA
            ...
FEATURES             Location/Qualifiers
     source          1..7067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   25..87
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        396..681
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        683..960
                     /label=chicken beta-actin promoter
     intron          961..1188
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1200..1209
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1209..1274
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1278..1298
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2598..2645
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(2673..2690)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2679..2886
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2887..3135
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2887..2907
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3058..3077
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   3664..3698
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        3756..4059
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4060..4263
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4214..4234
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4292..5002
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     DDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4437..4455)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    5019..5243
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5270..5374
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5375..5443,5444..6235)
                     /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(5593..5612)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6406..6994
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6895..6914
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatcccctga ttctctcgag ttaaccctag aaagataatc atattgtgac gtacgttaaa
       61 gataatcatg cgtaaaattg acgcatgtgt tttatcgatc tgtatatcga ggtttattta
      121 ttaatttgaa tagatattaa gttttattat atttacactt acatactaat aataaattca
      181 acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt
      241 ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact tattttgtac
      301 tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat acataataga
      361 aacaaattat acttattaat taatctagag gtacccgtta cataacttac ggtaaatggc
      421 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg
      481 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      541 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      601 tggcattgtg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      661 ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc
      721 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
      781 atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
      841 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      901 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      961 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
     1021 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
     1081 ccgggctgta attagctgag caagaggtaa gggtttaagg gatggttggt tggtggggta
     1141 ttaatgttta attacctgga gcacctgcct gaaatcactt tttttcaggt tggaccggtg
     1201 ccaccatgga ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag
     1261 acgatgacga taagatgcct aagaagaaga gaaaggtgat gggaaagggc gtgctggcca
     1321 aggtgatgaa atacgagctg agatacctgg atggttgtgg cgacttcagc aatatgcagg
     1381 agcaggtgtg ggccctgcag cggcagacac gggaaatcct gaacagatcc atccaaatcg
     1441 ccttccaatg gcgctgcgcc aacagcgagc accacagaaa gaccggcgag tacctggacc
     1501 tgaaaacgga aaccggctac aagagactgg atggccacat ctacaactgc ctgaagggcc
     1561 agtacgagga catggccaca tctaacctga acgccaccat ccagaaggct tggaagaagt
     1621 ataactccag caagaaggaa atcctgaggg gcagcatgag catccccagc tacaagatga
     1681 accagcctct gcgactggac aagaataccg tgaaactgtc tgagggcgag cggaacccaa
     1741 tcgtgaccct gacactgttt agcgacaagt tcaagcgggc ccagggcgtg tccaacgtga
     1801 agtttagcat gcctctgcac gacggcaccc agagagccat cttcgccaac ctgatgaacg
     1861 gcacctacca gctgggagag tgccagctgg tgtacaaacg gcctaagtgg ttcctgttcg
     1921 tgacatacaa gttccccccc gtggaacatc ctctcgatcc tgacaagatt ctgggcgtcg
     1981 acatgggcga ggcctgcgcg ctttatgcct ctacattcgg cgagcacggc tacctgaaga
     2041 tcgatggagg cgagattaca aagtacgcca agaagatgga agctagaatc cggagcatgc
     2101 agaagcaggc tgctcactgt ggcgaaggca gaatcgggca cggcaccaaa acaagagtgt
     2161 ctgtggtgta ccaggccaag gacaaggtgg ccagattcag agataccatc aaccacagat
     2221 actctaaggc cctgatcgac tacgccctga agaaccagtg tggcaccatc cagatggaag
     2281 atctgaccgg catcaaggaa gatacaggat ttccaaagtt cctgagacat tggacctact
     2341 acgacctgca gagcaagatc gaggctaagg ccgccgagca cggcatccaa gttgtcaaga
     2401 tcaaccctag acacaccagc cagcgctgca gcagatgtgg acacatcgac aaagccaata
     2461 gaaccagcca agctgatttc tgctgcacca agtgcggctt cagcgccaat gccgacttta
     2521 atgccagcca gaacatcagc atcagaaaca tcgacaagat tatcgccaag gctatcggcg
     2581 ccaaccggaa gcagaccaaa aggccggcgg ccacgaagaa ggccggccag gcaaagaaga
     2641 agaagtaaga attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     2701 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2761 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2821 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
     2881 tggggagagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     2941 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     3001 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     3061 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     3121 aaaggacgaa acaccgaggg ccgacttccc ggcccaaaat cgagacagta gccgtaaaac
     3181 gttgagtttc agcgtgggcg acacactcga aaaggttaag atatgcacat agtaatccgt
     3241 gcatgagccg cgaaagcggc ttgaaggcca ttacagtagg agcatacttt tatttttttg
     3301 cggccgcgtc gacggcgcgc cctagacgaa gcagctccag cctacacaat cgctcaagac
     3361 gtgtaatgct tttattatat attagtcacg atatctataa caagaaaata tatatataat
     3421 aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta
     3481 aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa
     3541 tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga
     3601 tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga
     3661 atgcatgcgt caattttacg cagactatct ttctagggtt aaggcaacgc ggccttttta
     3721 cggttcctgg ccttttgctg gccttttgct ctagacgtta cataacttac ggtaaatggc
     3781 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     3841 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     3901 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     3961 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     4021 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     4081 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     4141 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     4201 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     4261 gctggtttag tgaaccgtca gatccgctag catggtgagc aagggcgagg aggataacat
     4321 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     4381 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     4441 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     4501 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     4561 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgatgacg gcggcgtggt
     4621 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     4681 cggcaccaac ttcccctccg acggccccgt aatgcagaag aaaaccatgg gctgggaggc
     4741 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     4801 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     4861 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
     4921 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     4981 catggacgag ctgtacaagt aactcttggc ccggatccct gtgccttcta gttgccagcc
     5041 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     5101 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     5161 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaaaacaata gcaggcatgc
     5221 tggggatgcg gtgggctcta tggggatccg cacttttcgg ggaaatgtgc gcggaacccc
     5281 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5341 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5401 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5461 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5521 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5581 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5641 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5701 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5761 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5821 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5881 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5941 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6001 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6061 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6121 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6181 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6241 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6301 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6361 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6421 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6481 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6541 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6601 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6661 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6721 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6781 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6841 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6901 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6961 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7021 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgt
//
