LOCUS       sequence-440602-        8125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of NLS-rich iCas12a construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8125)
  AUTHORS   Chen K, Stahl EC, Kang MH, Xu B, Allen R, Trinidad M, Doudna JA
  TITLE     Engineering self-deliverable ribonucleoproteins for genome editing 
            in the brain.
  JOURNAL   Nat Commun. 2024 Feb 26;15(1):1727. doi: 10.1038/s41467-024-45998-2.
  PUBMED    38409124
REFERENCE   2  (bases 1 to 8125)
  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: "2024-02-26"; volume: "15(1):1727. doi"; pages: " 
            10.1038/s41467-024-45998-2"
COMMENT     Sequence Label: His_CL7_2NLS_iCas12a-4NLS
FEATURES             Location/Qualifiers
     source          1..8125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1762..1782
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1792..1812
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1822..1842
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1852..1872
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     3'UTR           1883..2027
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     terminator      2103..2174
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        2280..2384
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2385..2453,2454..3245)
                     /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(2603..2622)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3416..4004
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3905..3924
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4176..5258)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5220..5239
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5259..5336)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5436..5441,5442..5457,5458..5464)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5470..5486
                     /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)."
     5'UTR           5508..5641
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             5654..5671
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6083..6106
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6116..6136
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6146..6166
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 cgctacccta aatggtcaaa cgcgtacgat tttaatttct cagaaacaga gaaatacaaa
       61 gatattgcgg ggttttatcg cgaagtcgaa gaacaagggt ataaggtgtc gttcgaatcg
      121 gcttccaaaa aggaagtgga taaacttgtt gaagaaggca aactctatat gttccagatc
      181 tacaacaaag atttctccga caagagccat ggcacgccga acttacatac catgtacttt
      241 aaattactgt tcgatgaaaa taatcatggt cagattcgct tgagcggtgg cgccgaactg
      301 tttatgcgcc gcgcatcatt gaaaaaggaa gaacttgttg tgcacccagc aaacagtccg
      361 attgcaaaca aaaatccgga taacccgaag aaaactacca ccttaagcta tgatgtatat
      421 aaagataaac gttttagcga agatcagtat gaactccaca ttccgatcgc gattaacaaa
      481 tgtcctaaga acatctttaa aatcaatacc gaggttcgtg tactgctcaa acacgacgac
      541 aacccctatg ttattggcat tgaccgcggc gaacgcaacc tgctgtatat tgtcgttgtg
      601 gatggcaaag gtaacattgt tgaacagtat agccttaatg aaatcatcaa taatgtcaac
      661 ggcattcgta ttaaaacgga ctatcatagc ctgctggaca aaaaagagaa agaacgcttc
      721 gaagcccgtc agaattggac ctctatcgaa aatatcaagg aactgaaagc tgggtacatt
      781 tctcaagtgg tccacaagat ttgcgaactg gtggaaaagt atgacgcggt gatcgcactg
      841 gaggatctga attcgggctt taaaaatagc cgtgtgaagg tggagaaaca ggtttaccaa
      901 aagttcgaaa aaatgctgat taacaaactt aattacatgg tggacaaaaa atctaatcca
      961 tatgcgaccg gcggggctct caaaggctat cagattacga ataaattcga aagcttcaaa
     1021 tccatgtcta cccagaacgg atttatcttt tacatccccg cctggctgac ttccaaaatt
     1081 gatccgagta ccggttttgc taatctgctg aaaaccaaat atacgtcgat cgccgattca
     1141 aaaaaattca ttagcagctt cgaccgtatt atgtacgtcc cggaagaaga tctgttcgaa
     1201 ttcgcccttg actataaaaa tttctcgcgt acggatgccg attatatcaa aaaatggaaa
     1261 ctgtatagct acggcaaccg catccgcatt tttcgcaacc caaagaagaa caacgtattt
     1321 gactgggagg aagtttgctt gacgagcgct tacaaagagc tctttaacaa atatgggatc
     1381 aattatcagc taggggacat ccgtgtgttg ctgtgtgagc agtcagataa agcgttctac
     1441 agctctttca tggctttaat gacgctgatg ttgcagatgc gcaattcgat cactggccgc
     1501 accgatgtcg atttcctgat tagcccggtc aaaaatagcg atggcatttt ttacgattca
     1561 cgtaactatg aagcccagga aaacgctatt ctgccgaaga acgctgatgc gaacggcgcg
     1621 tacaatattg cccgtaaggt attgtgggca attggtcagt tcaagaaagc ggaagatgag
     1681 aagttggata aggtcaaaat tgcgatcccg aataaagaat ggctggaata tgcgcagacc
     1741 agcgtgaaac atggcggatc accgaagaag aaacgcaaag tcggaggtag ccctaagaag
     1801 aagcgtaagg ttggggggtc gccaaagaag aaacgtaaag ttgggggctc gccgaaaaag
     1861 aaacgcaagg tgtaataggt aatctctgct taaaagcaca gaatctaaga tccctgccat
     1921 ttggcgggga tttttttatt tgttttcagg aaataaataa tcgatcgcgt aataaaatct
     1981 attattattt ttgtgaagaa taaatttggg tgcaatgaga atgcgcaggc cctttcgtct
     2041 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg tcgagtaagg atctccaggc
     2101 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     2161 cggtgaacgc tctcgtcgac actatggttg ctttgacgta tgcggtgtga aataccgcac
     2221 agatgcgtaa ggagaaaata ccgcatcagg cgtcaggtgg cacttttcgg ggaaatgtgc
     2281 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2341 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2401 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2461 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2521 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2581 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     2641 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2701 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2761 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2821 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2881 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     2941 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3001 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3061 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3121 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3181 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3241 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3301 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3361 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3421 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3481 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3541 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     3601 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3661 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3721 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3781 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3841 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3901 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3961 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4021 cctttttacg gttcctggcc ttttgctggc cttttgctca catagtcatg ccccgcgccc
     4081 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4141 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4201 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4261 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4321 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4381 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4441 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4501 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4561 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4621 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4681 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4741 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4801 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4861 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4921 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4981 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5041 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5101 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     5161 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     5221 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     5281 tcttccgggc actatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     5341 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5401 gccgttgagc accgccgccg caaggaatgg tgtggttgac aattaatcat cggctcgtat
     5461 aatgtgtaat tgtgagcgga taacaatttg atgtgctagc gcatatccag tgtagtaagg
     5521 caagtccctt caagagttat cgttgatacc cctcgtagtg cacattcctt taacgcttca
     5581 aaatctgtaa agcacgccat atcgccgaaa ggcacactta attattaaga ggtaatacac
     5641 catgaaatct tctcaccatc accatcacca tggttctagc aaaagcaatg aaccgggtaa
     5701 agcaaccggt gaaggtaaac cggttaataa caaatggctg aacaatgccg gtaaagatct
     5761 gggtagtccg gttccggatc gtattgcaaa taaactgcgt gataaagaat tcgagagctt
     5821 cgatgatttt cgtgaaacct tttgggaaga agttagcaaa gatcctgaac tgagcaaaca
     5881 gtttagccgc aataacaatg atcgtatgaa agttggtaaa gcaccgaaaa cacgtaccca
     5941 ggatgttagc ggtaaacgta cctcatttga actgaatcat cagaaaccga ttgaacagaa
     6001 tggtggcgtt tatgatatgg ataacattag cgttgttacc ccgaaacgca acattgatat
     6061 tgaaggcggt ggtggtggtt ctttggaggt tttgttccag ggtccaaacg cgacaccaaa
     6121 gaaaaagcgt aaagtcgggg gatctccgaa aaagaaacgg aaagtaggta tccacggagt
     6181 cccagcagct accatgagca aactggagaa gttcacgaac tgttattctc tgtcgaaaac
     6241 cttacgtttt aaggccattc cggtaggtaa aacgcaagaa aatatcgaca ataaacgtct
     6301 actggttgaa gatgaaaagc gtgccgaaga ttataaaggt gttaaaaagc tgcttgatcg
     6361 ctattacctg tcttttatca acgatgttct gcattccatc aaattgaaaa acttgaataa
     6421 ttacatctcg ttatttcgca aaaaaacccg taccgaaaaa gaaaacaagg agctggaaaa
     6481 ccttgaaatt aatctgcgta aagaaatcgc gaaagcattt aaaggtaatg aaggatataa
     6541 atctctgttt aaaaaagaca ttattgaaac catcctgcct gagttcttgg atgataaaga
     6601 tgaaattgcc ctggttaata gctttaatgg ctttacgact gcatttaccg gcttttttga
     6661 taaccgtgaa aacatgttca gcgaagaagc gaaatcgact tccattgcct tccgttgcat
     6721 taatgaaaac ctgacccgct acatttctaa tatggacatc tttgaaaaag ttgatgccat
     6781 tttcgacaaa catgaagttc aagaaatcaa agaaaagatt ttaaactccg attatgatgt
     6841 tgaagatttc tttgagggag aatttttcaa ttttgttctc acccaagagg gtattgatgt
     6901 atataacgcc attatcggag gatttgttac ggaatcaggc gaaaagatta aaggactgaa
     6961 tgaatacatt aatttataca atcagaaaac caaacagaaa ttgccaaaat tcaaaccact
     7021 gtataaacag gtattaagcg accgtgaatc cttgtccttt tatggcgaag gctatacaag
     7081 cgatgaagag gtgctcgaag tattccgtaa tactttgaac aaaaactcgg aaatcttctc
     7141 cagcattaaa aaactggaaa aattgtttaa aaatttcgat gaatatagta gcgcaggcat
     7201 cttcgtcaaa aatggccctg cgatttcaac catttctaaa gacattttcg gtgaatggaa
     7261 cgtgatccgc gataaatgga acgcggagta tgacgacatt catctgaaaa aaaaagccgt
     7321 tgttaccgaa aagtatgaag atgaccgccg caagagcttt aaaaaaattg gtagtttctc
     7381 actggaacag ttacaagaat atgcagacgc cgatctgtcg gttgtggaaa agttaaaaga
     7441 gattattatc cagaaagtcg atgaaattta taaagtgtac ggctccagcg aaaagctctt
     7501 tgatgccgac tttgttttag aaaaatcgct gaaaaaaaac gatgccgtgg tggccatcat
     7561 gaaagatctg ttggattcgg tcaaatcgtt cgaaaactat atcaaggcgt tctttggcga
     7621 agggaaagaa accaatcgtg acgaatcgtt ctatggcgat tttgtgttgg cgtatgatat
     7681 tcttctgaaa gtagatcaca tctatgatgc tatccgcaac tacgtgaccc agaaaccgta
     7741 ttcgaaggat aaatttaagc tgtattttca aaaccctcag tttatgggtg gctgggataa
     7801 agataaagaa accgactacc gcgcgacgat cttgcgctat ggctctaagt actatttagc
     7861 catcatggat aaaaagtatg caaaatgcct ccagaaaatc gataaggatg atgtcaacgg
     7921 gaactacgaa aaaatcaact acaaactctt gcctggcccg aataaaatgc tgccgaaagt
     7981 ctttttttcc aaaaaatgga tggcctatta taacccttcc gaagatattc agaagattta
     8041 caagaacggt acctttaaaa aaggtgacat gtttaatctg aatgactgtc acaagcttat
     8101 tgatttcttc aaagatagta tttct
//
