LOCUS       sequence-464910-        9138 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses hiNLS variant t-M4 inside 0C triNLS-SpCas9-NGG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9138)
  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 9138)
  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: pEN017-SpCas9-hiNLS-t-M4
FEATURES             Location/Qualifiers
     source          1..9138
                     /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             6766..6792
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             6808..6834
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             7222..7242
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7258..7305
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(7435..7453)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7449..7496
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7862..7867,7868..7884,7885..7890))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7917..7935)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8003..8107
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8108..8176,8177..8968)
                     /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(8326..8345)
                     /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 tggagtagat gctaaagcga ttctttctgc acgattgagt
     3661 aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa aaatggctta
     3721 tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc aaattttgat
     3781 ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga tttagataat
     3841 ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa gaatttatca
     3901 gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa ggctccccta
     3961 tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct tttaaaagct
     4021 ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca atcaaaaaac
     4081 ggatatgcag gttatattga tgggggagct agccaagaag aattttataa atttatcaaa
     4141 ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa tcgtgaagat
     4201 ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat tcacttgggt
     4261 gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa agacaatcgt
     4321 gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc attggcgcgt
     4381 ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac cccatggaat
     4441 tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg catgacaaac
     4501 tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct ttatgagtat
     4561 tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat gcgaaaacca
     4621 gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa aacaaatcga
     4681 aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatc ttttgatagt
     4741 gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta ccatgatttg
     4801 ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga tatcttagag
     4861 gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga aagacttaaa
     4921 acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg ccgttatact
     4981 ggttggggac gtttgtctcg aaaattgatt aatggtatta gggataagca atctggcaaa
     5041 acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat gcagctgatc
     5101 catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc tggacaaggc
     5161 gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa aaaaggtatt
     5221 ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca taagccagaa
     5281 aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca gaaaaattcg
     5341 cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca gattcttaaa
     5401 gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta ttatctccaa
     5461 aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag tgattatgat
     5521 gtcgatcaca ttgttccaca aagtttcctt aaagacgatt caatagacaa taaggtctta
     5581 acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga agtagtcaaa
     5641 aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca acgtaagttt
     5701 gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc tggttttatc
     5761 aaacgccaat tggttgaaac tcgccaaatc actaagcatg tggcacaaat tttggatagt
     5821 cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa agtgattacc
     5881 ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa agtacgtgag
     5941 attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg aactgctttg
     6001 attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa agtttatgat
     6061 gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc aaaatatttc
     6121 ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa tggagagatt
     6181 cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg ggataaaggg
     6241 cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat tgtcaagaaa
     6301 acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag aaattcggac
     6361 aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt tgatagtcca
     6421 acggtagctt attcagtcct agttgttgct aaggtggaaa aagggaaatc gaagaagtta
     6481 aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt tgaaaaaaat
     6541 ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt aatcattaaa
     6601 ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct ggctagtgcc
     6661 ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa ttttttatat
     6721 ttagctagtc attatgaaaa gttgaagggt ggaagtggca gtggtccggc cgcgaagaaa
     6781 aagaaactgg acggcagcgg cagtggcccc gcggctaaga agaaaaagct ggatggaagc
     6841 ggcagcggga gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat
     6901 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat
     6961 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa
     7021 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt
     7081 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat
     7141 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag
     7201 ctaggaggtg acgccgggtc acccaagaag aagcgcaaag ttgacggatc gggatcaaaa
     7261 cgcccagccg caaccaagaa agcaggtcaa gctaaaaaaa agaagttgga atgataacat
     7321 tggaaatgga tcactgacat tgcggctcgt catcttgttg caggtcgcta tcacgcctgt
     7381 gcgttaatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     7441 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     7501 aaggaggaac tatatccgga tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg
     7561 atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg gtcggacagt
     7621 gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag cagcacgcca
     7681 tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg cagtaccggc
     7741 ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca
     7801 ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg
     7861 cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct
     7921 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     7981 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     8041 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataac attgaaaaag
     8101 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     8161 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     8221 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     8281 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     8341 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     8401 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     8461 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     8521 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     8581 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     8641 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     8701 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     8761 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     8821 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     8881 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     8941 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     9001 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     9061 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     9121 aaagatcaaa ggatcttc
//
