LOCUS       sequence-486531-        8683 bp    DNA     circular SYN 17-SEP-2025
DEFINITION  Mammalian expression of Flex-Cas12a with 2xSV40 NLS at N-terminus 
            and 2xSV40 at C-terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8683)
  AUTHORS   Ma E, Chen K, Shi H, Wasko KM, Esain-Garcia I, Trinidad MI, Zhou K, 
            Ye J, Doudna JA
  TITLE     Directed evolution expands CRISPR-Cas12a genome-editing capacity.
  JOURNAL   Nucleic Acids Res. 2025 Jul 8;53(13):gkaf649. doi: 
            10.1093/nar/gkaf649.
  PUBMED    40671523
REFERENCE   2  (bases 1 to 8683)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "2025-07-8"; volume: "53(13):gkaf649. doi"; 
            pages: " 10.1093/nar/gkaf649"
COMMENT     Sequence Label: pEM3_Euk_2NLS-Flex-Cas12a (LbCas12a)-2NLS
FEATURES             Location/Qualifiers
     source          1..8683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1167..1546
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1548..1825
                     /label=chicken beta-actin promoter
     intron          1826..2842
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2766..2788
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2850..2869
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2935..2944
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2947..2967
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2977..2997
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6691..6738
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6757..6777
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6867..6886)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6932..6987
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6986..7005)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(7348..7364)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7348..7364)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7361..7383)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7372..7388
                     /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)."
     promoter        complement(join(7396..7402,7403..7420,7421..7426))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7441..7462
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7521..7716
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7567..7702
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    7722..7856
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7759..7778)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7813..7832
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7924..7941)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8095..8683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8175..8194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgggtcgaca ttgattattg actagttatt aatagtaatc
     1201 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     1261 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     1321 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     1381 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     1441 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     1501 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
     1561 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
     1621 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg
     1681 cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa
     1741 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
     1801 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
     1861 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
     1921 gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct
     1981 tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg
     2041 ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc
     2101 cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag
     2161 tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg
     2221 gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc
     2281 ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc
     2341 gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg
     2401 gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg
     2461 gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct
     2521 tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc
     2581 gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc
     2641 cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct
     2701 ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag
     2761 ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca
     2821 tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca
     2881 ttttggcaaa gaattctgca gtcgacggta ccgcgggccc gggatccacc ggtcgccacc
     2941 atgggcccaa agaaaaagag gaaagtcggc agtggaccta aaaagaaacg aaaggttggg
     3001 tcaggttcta gcaagctgga aaaatttacc aactgttaca gcctgagcaa aaccctgagg
     3061 ttcaaggcca tccccgtggg caagacccaa gagaacatcg ataacaagag actgctggtc
     3121 gaggatgaga agcgtgcaga ggactacaag ggcgtgaaga agctcctgga tcggtactac
     3181 ctgagtttca tcaacgacgt gctgcacagc atcaagctga agaacctgaa caactacatc
     3241 agcctgttta gaaagaaaac cagaaccgaa aaagaaaata aggagctgga aaatctggaa
     3301 atcaacctga gaaaggaaat cgctaaggcc tttaagggaa acgaaggata caaatccctg
     3361 tttaagaaag acatcatcga gacaattctg ccagagttcc tggacgacaa agacgagata
     3421 gctcttgtga acagcttcaa caggttcacg accgccttca ccggattctt cgacaacagg
     3481 gaaaacatgt tcagtgagga agccaagtct accagcatcg ctttccggtg catcaatgag
     3541 aacctgaccg tgtatatctc taatatggat atctttgaaa aagtggacgc tattttcgac
     3601 aagcacgagg tgcaggagat caaggagaag atcctgaaca gcgactacga cgtggaggac
     3661 ttcttcgagg gcgaattttt caacttcgtg ctgacacaag agggcatcga cgtttacaac
     3721 gccatcatag gcggcttcgt gaccgagtcc ggcgaaaaga tcaagggcct taacgagtac
     3781 atcaacctgt ataaccagaa aaccaagcag aagctgccta agttcaagcc tctttataag
     3841 caggtcctga gcgaccggga aagcctgagc ttctacggcg agggctacac atctgacgag
     3901 gaagtgctgg aggtctttcg gaataccctg aacaagaact ctgagatctt cagctctatt
     3961 aagaaactgg aaaagctgtt caagaatttc gacgagtata gtagcgccgg gatcttcgtg
     4021 aagaacggcc ctgccattag caccatcagc aaggacatct tcggcgaatg gaacgttatc
     4081 cgcgataagt ggaacgccga gtacgatgac atccacctga aaaagaaggc cgtggtgacc
     4141 gagaagtacg aagatgacag aagaaagagc ttcaaaaaga tcggctcatt tagcctggaa
     4201 cagctgcagg agtacgccga cgccgacctg tccgtggtcg aaaagctgaa ggaaattatc
     4261 atccaaaaag tggacgagat ctataaggtg tacgggagct ctgagaagct gttcgacgcc
     4321 gactttgtgc tggaaaagtc actgaaaaag aacgacgctg tggtcgccat catgaaggat
     4381 ctgctggact ccgtgaagag cttcgagaac tacatcaagg ccttcttcgg cgaaggcaag
     4441 gagaccaacc gggacgagtc tttttacggc gatttcgtgc tggcctacga catcctgctg
     4501 aaggtggacc acatctacga cgccatcaga aactacgtga cccagaagcc ctacagcaaa
     4561 gacaagttta agctgtactt ccagaatcct cagttcatgg gaggatgggg caaagacaag
     4621 gaaaccgact accgggctac aattctgaga tacggcttta agtactacct tgcaatcatg
     4681 gacaaaaagt atgccaagtg cctgcagaaa attgataagg atgacgtgaa cggcaactac
     4741 gagaagatca actacaagct cctgcccggc cccaacaaga tgctgccaaa ggtgttcttc
     4801 tccaaaaagt ggatggccta ttacaaccct tctgaggata tccagaagat ctataagaac
     4861 ggaacattca agaagggcga tatgttcaac ctgaatgact gccacaagct gatcgacttc
     4921 tttaaagaca gcatctccag ataccctaag tggtccaacg cctacgactt caacttcagc
     4981 gaaacagaaa agtacaagaa catcgccggc ttctacagag aggtggaaga acaaggctac
     5041 aaggtcagct tcgagagcgc tagcaagaag gaggtggaca agctggtgga ggagggaaag
     5101 ctgtacatgt tccagatcta caacaaggat ttcagcgaca agagccacgg cacacctaac
     5161 ctgcacacca tgtatttcaa gctgctgttt gacgagaaca accatggcca gatccggctg
     5221 tctggcggcg ccgaactgtt catgcggaga gcctctttga agaaggaaga gctggtggtg
     5281 caccctgcca atagccctat cgccaacaag aacccagata acccaaagaa aaccaccaca
     5341 ctgagctacg acgtgtacaa agataagaga ttcagcgagg atcagtacca gctgcatatc
     5401 cctatcgcca tcaataagtg tcctaaaaac atcttcaaga tcaataccga ggtgcgggtg
     5461 ttgctgaagc acgacgataa cccttacgtg atcggaatcg acaggggcga gagaaacctg
     5521 ctgtacatcg tggtggtgga cggcaagggc aatatcgtgg agcaatacag cctgaatgag
     5581 atcatcaaca atttcaacgg catccgcata aaaacagatt accactctct gctggataag
     5641 aaagagaaag agcggtttga ggccagacag aactggacca gcatcgagaa tatcaaagag
     5701 ttaaaggccg gctacatcag ccaggtggtg cacaaaatct gcgagctggt ggaaaagtac
     5761 gacgctgtga tcgcccttga ggatctaaac agcggcttta agaacagcag agtgaaggta
     5821 gaaaagcagg tgtatcagaa gttcgagaag atgctcatcg ataagctgaa ctacatggtg
     5881 gataagaaaa gcaacccctg cgccacaggc ggagctctga agggctacca gatcaccaac
     5941 aaattcgaga gctttaagag catgtcgacc cagaatggtt tcatctttta catccccgcc
     6001 tggctcacct caaagatcga tcccagcaca ggcttcgtca acctgctgaa aaccaagtac
     6061 accagcatcg ccgatagcaa gaagtttatc tcgtccttcg acagaatcat gtacgtgccc
     6121 gaggaggacc tgttcgagtt cgccctggac tacaagaact tctcccggac agatgccgac
     6181 tacattaaga agtggaagct gtacagctac ggaaacagga tcagaatctt tagaaacccc
     6241 aaaaagaaca acgtattcga ttgggaggaa gtgtgcctga cctcggccta caaggagctg
     6301 ttcaacaagt acggcatcaa ctaccagcag ggcgacatcc gggccctgct ctgtgaacag
     6361 agcgacaagg ctttctacag cagcttcatg gccctgatga gcctgatgct gcagatgaga
     6421 aacagcatca caggaagaac agacgtggac tttctgatct ctcctgtgaa gaacagcgac
     6481 ggcatcttct acgacagcag aaactatgaa gcccaggaga acgcgatcct gcctaagaac
     6541 gcagacgcca acggcgccta caatatcgcc agaaaggtgc tgtgggccat tggccagttc
     6601 aagaaggctg aagatgagaa actggataaa gtgaagatcg ccatctccaa caaggaatgg
     6661 ctggaatacg cccagaccag cgtgaagcac aaaaggccgg cggccacgaa aaaggccggc
     6721 caggcaaaaa agaaaaaggg atcctctggt ggttctccca agaagaagag gaaagtctaa
     6781 agcggccgca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat
     6841 gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc
     6901 atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata
     6961 gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta
     7021 aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc
     7081 atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat
     7141 tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg
     7201 ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc
     7261 ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag atccctcgac
     7321 ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     7381 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     7441 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     7501 cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     7561 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     7621 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     7681 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt
     7741 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     7801 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct
     7861 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     7921 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     7981 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     8041 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     8101 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     8161 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     8221 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     8281 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     8341 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     8401 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     8461 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     8521 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     8581 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     8641 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caa
//
