LOCUS       sequence-296112-        6322 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of APEX2-NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6322)
  AUTHORS   Kurihara M, Kato K, Sanbo C, Shigenobu S, Ohkawa Y, Fuchigami T, 
            Miyanari Y
  TITLE     Genomic Profiling by ALaP-Seq Reveals Transcriptional Regulation by 
            PML Bodies through DNMT3A Exclusion.
  JOURNAL   Mol Cell. 2020 May 7;78(3):493-505.e8. doi: 
            10.1016/j.molcel.2020.04.004. Epub 2020 Apr 29.
  PUBMED    32353257
REFERENCE   2  (bases 1 to 6322)
  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: 
            "10.1016/j.molcel.2020.04.004"; journalName: "Mol Cell"; date: 
            "2020-05-7- 7"; volume: "78"; issue: "3"; pages: "493-505.e8"
COMMENT     Sequence Label: pEF-APEX2-NLS
FEATURES             Location/Qualifiers
     source          1..6322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1187..1196
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1196..1219
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2012..2032
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2036..2056
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2060..2080
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    join(2126..2700,2701..2703,2704..2712)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2293..2310)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2520..2539
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2713..3312
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2713..2732)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3209..3229
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3390..3445
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3444..3463)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3806..3822)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3806..3822)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3819..3841)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3830..3846
                     /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(3854..3860,3861..3878,3879..3884))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3899..3920
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3979..4174
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4025..4160
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4180..4314
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4217..4236)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4271..4290
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4382..4399)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4553..5141)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4633..4652)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5312..6103,6104..6172))
                     /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     5935..5954
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6173..6277)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag ctaggcgcca ccatggatta
     1201 caaggatgac gacgataagg gcctgcttag cggccgcgga tccggcaaat catatcctac
     1261 agtgtctgcc gactatcagg acgctgtgga gaaagctaag aagaaactga gaggcttcat
     1321 cgctgagaag cggtgtgccc ccctgatgct gagactggct ttccacagcg ccggaacctt
     1381 cgacaaggga accaagacag gcggcccttt cggaaccatc aagcaccccg ctgagctcgc
     1441 ccatagcgct aacaacggac tggacatcgc cgtgcggctg ctggagcctc tgaaggccga
     1501 gttccctatc ctgagctatg ccgacttcta ccagctggct ggagtggtgg ccgtggaggt
     1561 taccggagga cctaaggtgc ccttccaccc tggaagagag gacaagcctg agcctccacc
     1621 cgagggacgg ctgcctgatc ctaccaaggg aagcgaccat ctgcgggacg tgttcggaaa
     1681 ggccatggga ctgaccgatc aagacatcgt ggccctcagc ggcggacata caatcggagc
     1741 tgctcacaag gagagaagcg gcttcgaagg cccctggaca agcaaccccc tgatcttcga
     1801 caacagctac tttaccgaac tgctcagcgg cgagaaggag ggactgttgc agctgcccag
     1861 cgacaaggct ctgctcagcg accctgtgtt cagacccctg gtggacaagt acgctgctga
     1921 cgaggatgcc ttcttcgctg actacgccga ggctcaccag aagctgagcg agctgggctt
     1981 cgctgacgct aagctcagat ctcgagctga tccaaaaaag aagagaaagg tagatccaaa
     2041 aaagaagaga aaggtagatc caaaaaagaa gagaaaggta ggatccaccg gatctagcca
     2101 tggctaactc gaggcccggg atccgcccct ctccctcccc cccccctaac gttactggcc
     2161 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     2221 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     2281 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
     2341 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     2401 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     2461 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     2521 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     2581 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa
     2641 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat
     2701 atggccacaa ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     2761 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     2821 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     2881 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgccgt ggcggtctgg
     2941 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     3001 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     3061 cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc
     3121 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     3181 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     3241 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     3301 cccggtgcct gaacgcgtta agtcgatttt tccctctgcc aaaaattatg gggacatcat
     3361 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
     3421 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
     3481 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat
     3541 gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc
     3601 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt
     3661 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct
     3721 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct
     3781 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3841 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3901 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3961 tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     4021 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4081 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4141 aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat
     4201 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4261 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc
     4321 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4381 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4441 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4501 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4561 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4621 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4681 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4741 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4801 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4861 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4921 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4981 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5041 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5101 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5161 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5221 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5281 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5341 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5401 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5461 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5521 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5581 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5641 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5701 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5761 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5821 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5881 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5941 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6001 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6061 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6121 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6181 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6241 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6301 ctgggtcgac attgattatt ga
//
