LOCUS       sequence-375498-        4722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses the bpNLS-linked DddIA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4722)
  AUTHORS   Lei Z, Meng H, Liu L, Zhao H, Rao X, Yan Y, Wu H, Liu M, He A, Yi C
  TITLE     Mitochondrial base editor induces substantial nuclear off-target 
            mutations.
  JOURNAL   Nature. 2022 May 12. pii: 10.1038/s41586-022-04836-5. doi: 
            10.1038/s41586-022-04836-5.
  PUBMED    35551512
REFERENCE   2  (bases 1 to 4722)
  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: "Nature. 
            2022 May 12. pii: 10.1038/s41586-022-04836-5. doi: 
            10.1038/s41586-022-04836-5."
COMMENT     Sequence Label: bpNLS-DddIA
FEATURES             Location/Qualifiers
     source          1..4722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             892..912
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             922..978
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(979..981,982..984,985..1695)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(979..981,982..984,985..1695)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1024..1045)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1285..1304)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1632..1653
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1708..1728
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1737..1754
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1777..1794)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1783..2007
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2078..2094)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2078..2094)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2091..2113)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2102..2118
                     /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(2126..2132,2133..2150,2151..2156))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2171..2192
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2309..2326)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2480..3068)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2560..2579)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3239..4030,4031..4099))
                     /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     3862..3881
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4100..4204)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4283..4302)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(4474..4722,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcgaagg aagcggatct
      481 atgtacgccg atgacttcga cggcgagatt gaaatcgacg aggtggacag cctggtcgaa
      541 ttcctgtcta gacggcctgc ctttgatgcc aacaacttcg tgctgacatt cgaggaaagc
      601 ggattccccc agctgaacat cttcgccaag aatgacatcg ccgtggttta ctatatggac
      661 atcggcgaga atttcgtgtc caaaggcaac agcgccagcg gaggcaccga gaagttctac
      721 gagaacaagc tgggcggcga agtggatctg agcaaggact gcgtggtgtc taaggaacag
      781 atgatcgagg ccgctaaaca atttttcgct acaaagcaga gacctgagca gctcacctgg
      841 agcgagctgt ctggcggctc aaaaagaacc gccgacggca gcgaattcga gcccaagaag
      901 aagaggaaag tcggaagcgg agctactaac ttcagcctgc tgaagcaggc tggagacgtg
      961 gaggagaacc ctggacctat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1021 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1081 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1141 cccgtgccct ggcccaccct cgtgaccacc ctgacctatg gagtgcagtg cttcagccgc
     1201 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1261 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1321 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1381 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1441 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1501 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1561 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1621 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1681 gacgagctgt acaagtctgg tggttctccc aagaagaaga ggaaagtcta accggtcatc
     1741 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     1801 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     1861 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     1921 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     1981 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     2041 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2101 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2161 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2221 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2281 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2341 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2401 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2461 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2521 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2581 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2641 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2701 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2761 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2821 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2881 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2941 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3001 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3061 gatctcaaga agatcctttg atcttttcta cggggtctga cactcagtgg aacgaaaact
     3121 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3181 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3241 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3301 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3361 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3421 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3481 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3541 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3601 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3661 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3721 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3781 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3841 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3901 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3961 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4021 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4081 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4141 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4201 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
     4261 atcccctagg gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     4321 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     4381 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     4441 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
     4501 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     4561 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     4621 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     4681 atttacggta aactgcccac ttggcagtac atcaagtgta tc
//
