LOCUS       sequence-367856-        7948 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express FLAG epitope and APEX2-tagged LMNA fusion protein in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7948)
  AUTHORS   Barutcu AR, Wu M, Braunschweig U, Dyakov BJA, Luo Z, Turner KM, 
            Durbic T, Lin ZY, Weatheritt RJ, Maass PG, Gingras AC, Blencowe BJ
  TITLE     Systematic mapping of nuclear domain-associated transcripts reveals 
            speckles and lamina as hubs of functionally distinct retained 
            introns.
  JOURNAL   Mol Cell. 2022 Mar 3;82(5):1035-1052.e9. doi: 
            10.1016/j.molcel.2021.12.010. Epub 2022 Feb 18.
  PUBMED    35182477
REFERENCE   2  (bases 1 to 7948)
  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.2021.12.010"; journalName: "Mol Cell"; date: 
            "2022-03-3- 3"; volume: "82"; issue: "5"; pages: "1035-1052.e9"
COMMENT     Sequence Label: pcDNA5-FRT/TO-3*Flag-APEX2 N-term LMNA
FEATURES             Location/Qualifiers
     source          1..7948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1034..1057
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1058..1804
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     protein_bind    1805..1829
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3834..3858)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3900..3917)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3906..4130
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4263..4282)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4414..4461
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4469..5491
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5621..5742
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5658..5677)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5712..5731
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5791..5807)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5791..5807)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5804..5826)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5815..5831
                     /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(5839..5845,5846..5863,5864..5869))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5884..5905
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6022..6039)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6193..6781)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6273..6292)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6952..7743,7744..7812))
                     /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     7575..7594
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7813..7917)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttccaccat ggactacaaa gaccatgacg gtgatacata
     1021 tgatcatgac atcgattaca aggatgacga tgacaaggga aagtcttacc caactgtgag
     1081 tgctgattac caggacgccg ttgagaaggc gaagaagaag ctcagaggct tcatcgctga
     1141 gaagagatgc gctcctctaa tgctccgttt ggcattccac tctgctggaa cctttgacaa
     1201 gggcacgaag accggtggac ccttcggaac catcaagcac cctgccgaac tggctcacag
     1261 cgctaacaac ggtcttgaca tcgctgttag gcttttggag ccactcaagg cggagttccc
     1321 tattttgagc tacgccgatt tctaccagtt ggctggcgtt gttgccgttg aggtcacggg
     1381 tggacctaag gttccattcc accctggaag agaggacaag cctgagccac caccagaggg
     1441 tcgcttgcct gatcccacta agggttctga ccatttgaga gatgtgtttg gcaaagctat
     1501 ggggcttact gaccaagata tcgttgctct atctgggggt cacactattg gagctgcaca
     1561 caaggagcgt tctggatttg agggtccctg gacctctaat cctcttattt tcgacaactc
     1621 atacttcacg gagttgttga gtggtgagaa ggaaggtctc cttcagctac cttctgacaa
     1681 ggctcttttg tctgaccctg tattccgccc tctcgttgat aaatatgcag cggacgaaga
     1741 tgccttcttt gctgattacg ctgaggctca ccaaaagctt tccgagcttg ggtttgctga
     1801 tgccacaagt ttgtacaaaa aagcaggctc caccatggag accccgtccc agcggcgcgc
     1861 cacccgcagc ggggcgcagg ccagctccac tccgctgtcg cccacccgca tcacccggct
     1921 gcaggagaag gaggacctgc aggagctcaa tgatcgcttg gcggtctaca tcgaccgtgt
     1981 gcgctcgctg gaaacggaga acgcagggct gcgccttcgc atcaccgagt ctgaagaggt
     2041 ggtcagccgc gaggtgtccg gcatcaaggc cgcctacgag gccgagctcg gggatgcccg
     2101 caagaccctt gactcagtag ccaaggagcg cgcccgcctg cagctggagc tgagcaaagt
     2161 gcgtgaggag tttaaggagc tgaaagcgcg caataccaag aaggagggtg acctgatagc
     2221 tgctcaggct cggctgaagg acctggaggc tctgctgaac tccaaggagg ccgcactgag
     2281 cactgctctc agtgagaagc gcacgctgga gggcgagctg catgatctgc ggggccaggt
     2341 ggccaagctt gaggcagccc taggtgaggc caagaagcaa cttcaggatg agatgctgcg
     2401 gcgggtggat gctgagaaca ggctgcagac catgaaggag gaactggact tccagaagaa
     2461 catctacagt gaggagctgc gtgagaccaa gcgccgtcat gagacccgac tggtggagat
     2521 tgacaatggg aagcagcgtg agtttgagag ccggctggcg gatgcgctgc aggaactgcg
     2581 ggcccagcat gaggaccagg tggagcagta taagaaggag ctggagaaga cttattctgc
     2641 caagctggac aatgccaggc agtctgctga gaggaacagc aacctggtgg gggctgccca
     2701 cgaggagctg cagcagtcgc gcatccgcat cgacagcctc tctgcccagc tcagccagct
     2761 ccagaagcag ctggcagcca aggaggcgaa gcttcgagac ctggaggact cactggcccg
     2821 tgagcgggac accagccggc ggctgctggc ggaaaaggag cgggagatgg ccgagatgcg
     2881 ggcaaggatg cagcagcagc tggacgagta ccaggagctt ctggacatca agctggccct
     2941 ggacatggag atccacgcct accgcaagct cttggagggc gaggaggaga ggctacgcct
     3001 gtcccccagc cctacctcgc agcgcagccg tggccgtgct tcctctcact catcccagac
     3061 acagggtggg ggcagcgtca ccaaaaagcg caaactggag tccactgaga gccgcagcag
     3121 cttctcacag cacgcacgca ctagcgggcg cgtggccgtg gaggaggtgg atgaggaggg
     3181 caagtttgtc cggctgcgca acaagtccaa tgaggaccag tccatgggca attggcagat
     3241 caagcgccag aatggagatg atcccttgct gacttaccgg ttcccaccaa agttcaccct
     3301 gaaggctggg caggtggtga cgatctgggc tgcaggagct ggggccaccc acagcccccc
     3361 taccgacctg gtgtggaagg cacagaacac ctggggctgc gggaacagcc tgcgtacggc
     3421 tctcatcaac tccactgggg aagaagtggc catgcgcaag ctggtgcgct cagtgactgt
     3481 ggttgaggac gacgaggatg aggatggaga tgacctgctc catcaccacc acggctccca
     3541 ctgcagcagc tcgggggacc ccgctgagta caacctgcgc tcgcgcaccg tgctgtgcgg
     3601 gacctgcggg cagcctgccg acaaggcatc tgccagcggc tcaggagccc aggtgggcgg
     3661 acccatctcc tctggctctt ctgcctccag tgtcacggtc actcgcagct accgcagtgt
     3721 ggggggcagt gggggtggca gcttcgggga caatctggtc acccgctcct acctcctggg
     3781 caactccagc ccccgaaccc agagccccca gaactgcagc atcatgtgaa tccacccagc
     3841 tttcttgtac aaagtggtct gactcgagtc tagagggccc gtttaaaccc gctgatcagc
     3901 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3961 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     4021 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     4081 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     4141 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     4201 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     4261 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4321 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4381 aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc
     4441 tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga
     4501 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     4561 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     4621 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     4681 cccgattccg gaagtgcttg acattgggga gttcagcgag agcctgacct attgcatctc
     4741 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     4801 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     4861 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     4921 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     4981 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     5041 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     5101 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     5161 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     5221 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     5281 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     5341 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     5401 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     5461 ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc
     5521 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5581 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5641 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5701 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5761 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5821 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5881 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5941 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6001 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     6061 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     6121 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     6181 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6241 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6301 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6361 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6421 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6481 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6541 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6601 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6661 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6721 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6781 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6841 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6901 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6961 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     7021 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     7081 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     7141 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     7201 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7261 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7321 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7381 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7441 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7501 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7561 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7621 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7681 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7741 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7801 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7861 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7921 atttccccga aaagtgccac ctgacgtc
//
