LOCUS       sequence-464908-        9237 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses hiNLS variant t-M1M4 inside 0C triNLS-SpCas9-NGG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9237)
  AUTHORS   Noel EA, Sahu SU, Wyman SK, Krishnappa N, Jeans C, Wilson RC
  TITLE     Hairpin Internal Nuclear Localization Signals in CRISPR-Cas9 Enhance
            Editing in Primary Human Lymphocytes.
  JOURNAL   CRISPR J. 2025 Apr;8(2):105-119. doi: 10.1089/crispr.2024.0080. Epub
            2025 Mar 31.
  PUBMED    40163415
REFERENCE   2  (bases 1 to 9237)
  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"; doi: 
            "10.1089/crispr.2024.0080"; journalName: "CRISPR J"; date: 
            "2025-04"; volume: "8"; issue: "2"; pages: "105-119"
COMMENT     Sequence Label: pEN019-SpCas9-hiNLS-t-M1M4
FEATURES             Location/Qualifiers
     source          1..9237
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2665..2684
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2768..2787
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2768..2786
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2875..2897
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2917..2934
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2944..2964
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2974..3000
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             3637..3663
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             3679..3705
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             6865..6891
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             6907..6933
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             7321..7341
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7357..7404
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(7534..7552)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7548..7595
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7961..7966,7967..7983,7984..7989))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(8016..8034)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8102..8206
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8207..8275,8276..9067)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(8425..8444)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     1681 acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata
     1741 tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa
     1801 ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc
     1861 ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac
     1921 aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag
     1981 cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg
     2041 atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc
     2101 ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa
     2161 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     2221 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     2281 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     2341 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     2401 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     2461 catcggtcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     2521 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     2581 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     2641 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     2701 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     2761 gcgaaattaa tacgactcac tatagggaga ccacaacggt ttccctctag tgccggctcc
     2821 ggagagctct ttaattaagc ggccgccctg caggactcga gttctagaaa taattttgtt
     2881 taactttaag aaggagatat acatatgaaa tcttctcacc atcaccatca ccatggttct
     2941 tctgaaaacc tgtacttcca atcccgcgca gcccccgcag ctaaaaagaa gaagttggat
     3001 aacgcgacat ctgataagaa atactcaata ggcttagata tcggcacaaa tagcgtcgga
     3061 tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt tctgggaaat
     3121 acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga cagtggagag
     3181 acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg tcggaagaat
     3241 cgtatttctt atctacagga gattttttca aatgagatgg cgaaagtaga tgatagtttc
     3301 tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga acgtcatcct
     3361 atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac tatctatcat
     3421 ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat ctatttggcc
     3481 ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt aaatcctgat
     3541 aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca attatttgaa
     3601 gaaaacccta ttaacgcaag tggaagtggt tcagggcccg ctgcaaaaaa gaagaaattg
     3661 gacggctccg gcagcggtcc ggctgcgaaa aaaaagaagc tggatggtag cggttctggc
     3721 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
     3781 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
     3841 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
     3901 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
     3961 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
     4021 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
     4081 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
     4141 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
     4201 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
     4261 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
     4321 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
     4381 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
     4441 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
     4501 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
     4561 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat
     4621 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg
     4681 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag
     4741 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta
     4801 aaagaagatt atttcaaaaa aatagaatct tttgatagtg ttgaaatttc aggagttgaa
     4861 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa
     4921 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc
     4981 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat
     5041 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga
     5101 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa
     5161 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt
     5221 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt
     5281 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt
     5341 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca
     5401 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc
     5461 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact
     5521 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg
     5581 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatcacat tgttccacaa
     5641 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt
     5701 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga
     5761 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa
     5821 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact
     5881 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat
     5941 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct
     6001 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc
     6061 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt
     6121 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag
     6181 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac
     6241 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa
     6301 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc
     6361 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga
     6421 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa
     6481 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta
     6541 gttgttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta
     6601 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct
     6661 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt
     6721 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat
     6781 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag
     6841 ttgaagggtg gaagtggcag tggtccggcc gcgaagaaaa agaaactgga cggcagcggc
     6901 agtggccccg cggctaagaa gaaaaagctg gatggaagcg gcagcgggag tccagaagat
     6961 aacgaacaaa aacaattgtt tgtggagcag cataagcatt atttagatga gattattgag
     7021 caaatcagtg aattttctaa gcgtgttatt ttagcagatg ccaatttaga taaagttctt
     7081 agtgcatata acaaacatag agacaaacca atacgtgaac aagcagaaaa tattattcat
     7141 ttatttacgt tgacgaatct tggagctccc gctgctttta aatattttga tacaacaatt
     7201 gatcgtaaac gatatacgtc tacaaaagaa gttttagatg ccactcttat ccatcaatcc
     7261 atcactggtc tttatgaaac acgcattgat ttgagtcagc taggaggtga cgccgggtca
     7321 cccaagaaga agcgcaaagt tgacggatcg ggatcaaaac gcccagccgc aaccaagaaa
     7381 gcaggtcaag ctaaaaaaaa gaagttggaa tgataacatt ggaaatggat cactgacatt
     7441 gcggctcgtc atcttgttgc aggtcgctat cacgcctgtg cgttaatccg gctgctaaca
     7501 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     7561 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     7621 atccacagga cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa
     7681 gcgagcagga ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat
     7741 agaaattgca tcaacgcata tagcgctagc agcacgccat agtgactggc gatgctgtcg
     7801 gaatggacga tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca
     7861 gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt
     7921 gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata
     7981 agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     8041 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     8101 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     8161 acaataaccc tgataaatgc ttcaataaca ttgaaaaagg aagagtatga gtattcaaca
     8221 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     8281 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     8341 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     8401 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     8461 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     8521 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     8581 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     8641 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     8701 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     8761 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     8821 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     8881 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     8941 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     9001 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     9061 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     9121 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     9181 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
