LOCUS       sequence-388964-        7024 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Vector encoding a human codon-optimized enRhCas12f1 driven by CBh 
            promoter, guide RNAs compatible with enRhCas12f1 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 7024)
  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 7024)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-enRhCas12f1-NLS_pA_phU6_gRNA
            ...
FEATURES             Location/Qualifiers
     source          1..7024
                     /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             2544..2591
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(2619..2636)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2625..2832
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2833..3081
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2833..2853
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3004..3023
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   3621..3655
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        3713..4016
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4017..4220
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4171..4191
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4249..4959
                     /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"
     polyA_signal    4976..5200
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5227..5331
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5332..5400,5401..6192)
                     /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(5550..5569)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6363..6951
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6852..6871
                     /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 ccrggsgggg sggggsgggg sgrggggsgg
      841 ggsggggsgr ggcggagagg kgcggcggca 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 gattacagtg cggaagctga
     1321 agattctgat cgacggcgag agcaggaacg agagctacaa gttcatccgg gacagcatgt
     1381 acgcccagta cctggccctg aacaaggcca tgagctacct gggtaccgcc tacctgagca
     1441 gagataagga gatcttcaag gaggccatca agtctctcaa caactctaat cctatcttcg
     1501 acaacattaa cttcggcaag ggaatcgata ccaagagcag cgtgaaccag acagtgaaga
     1561 aacacatcca ggccgacatt aagaatggcc tggccaaggg cgaaagatcc atccggaact
     1621 acaagcgtga ctaccccctg atgaccagag gcagagatct gaagtttttc tactgcgaca
     1681 ccaatagcac caaggtgaag gtgaaatggg tgaacggcat catctttgac gtgatgctgg
     1741 gaaaggaata caacaagaat gatctagagc tgagatcttt tctgaacaga gtcatcaaca
     1801 aggaatataa gatctcccaa agcagcatct gcttcgacaa gcacaacaga ctgatcctga
     1861 atctgagcgt gaacatcacc gacaacatcc ccaacgaggt ggtgaagggc agaattgtgg
     1921 gcgtcgacct gggcatgaaa atcccagctt atgtgacact gaacgacagc gagtacatcg
     1981 gaaaacctat cggcgacatc aacgacttcc tgaaggtgcg gaagcagttc aaagaaagaa
     2041 aggagcggct gcagaagcag ctggccatca acaagggcgg cagaggcatc accaacaaga
     2101 tgcagcggat ggacgccttc accaacaagg aaaagaactt cgctaatacc tacaaccacg
     2161 gcgtttctaa ggcaatcatc aactttgcta agaagtacaa ggccgagcag atcaacgtgg
     2221 agttcctggc tctggccggc agcgagaagg aaatcctgag ctccacaatc cgctactggt
     2281 cctactatca actgcaacag atgatcgagt acaaggccaa ccgggaaggc atcgccgtga
     2341 agtacgtgga cccttacctg acctcacaga cctgctgcaa gtgcggcaac tacgaggtgg
     2401 gacagagaat caaccaggag ctgttcgagt gtaaactgtg tggcaataaa atgaatgccg
     2461 atagaaacgc cagcttcaac atcgccagaa gcacaaagta catcagctct aaagaggaaa
     2521 gcgacttcta caaacagctc aaaaaaaggc cggcggccac gaagaaggcc ggccaggcaa
     2581 agaagaagaa gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     2641 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2701 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2761 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag
     2821 gcatgctggg gagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     2881 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     2941 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     3001 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     3061 ttgtggaaag gacgaaacac cgggacggct gatttagcag ccgaagtctg agggcatgta
     3121 gaaaaaagta taggtatata ccaacatact tgcattgcca ctcggaaagg gttaaccttg
     3181 gtcattgtgt taccgaccaa gcattccaga aatggaatgt aaatccatta cagtaggagc
     3241 atacttttat ttttttgcgg ccgcgtcgac ggcgcgccct agacgaagca gctccagcct
     3301 acacaatcgc tcaagacgtg taatgctttt attatatatt agtcacgata tctataacaa
     3361 gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa tataattatc
     3421 gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg
     3481 gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct
     3541 ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag
     3601 agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaag
     3661 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcta gacgttacat
     3721 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     3781 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     3841 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     3901 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     3961 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     4021 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     4081 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     4141 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     4201 aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcat ggtgagcaag
     4261 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     4321 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     4381 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     4441 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     4501 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     4561 gatgacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     4621 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaaa
     4681 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     4741 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     4801 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     4861 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     4921 cgccactcca ccggcggcat ggacgagctg tacaagtaac tcttggcccg gatccctgtg
     4981 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     5041 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     5101 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     5161 aacaatagca ggcatgctgg ggatgcggtg ggctctatgg ggatccgcac ttttcgggga
     5221 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5281 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5341 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5401 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5461 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5521 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5581 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5641 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5701 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5761 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5821 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5881 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5941 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6001 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     6061 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6121 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6181 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6241 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6301 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6361 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6421 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6481 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6541 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6601 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6661 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6721 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6781 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6841 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6901 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6961 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     7021 gcgt
//
