LOCUS       sequence-367946-        7388 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express FLAG epitope and APEX2-tagged SP100 fusion protein in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7388)
  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 7388)
  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 C-term SP100
FEATURES             Location/Qualifiers
     source          1..7388
                     /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"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     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"
     protein_bind    987..1011
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2452..2476)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2487..3233
                     /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"
     CDS             3234..3299
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3340..3357)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3346..3570
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3703..3722)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3854..3901
                     /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             3909..4931
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5061..5182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5098..5117)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5152..5171
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5231..5247)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5231..5247)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5244..5266)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5255..5271
                     /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(5279..5285,5286..5303,5304..5309))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5324..5345
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5462..5479)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5633..6221)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5713..5732)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6392..7183,7184..7252))
                     /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     7015..7034
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7253..7357)
                     /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 ctttagacaa gtttgtacaa aaaagttggc atggcaggtg
     1021 ggggcggcga cctgagcacc aggaggctga atgaatgtat ttcaccagta gcaaatgaga
     1081 tgaaccatct tcctgcacac agccacgatt tgcaaaggat gttcacggaa gaccagggtg
     1141 tagatgacag gctgctctat gacattgtat tcaagcactt caaaagaaat aaggtggaga
     1201 tttcaaatgc aataaaaaag acatttccat tcctcgaggg cctccgtgat cgtgatctca
     1261 tcacaaataa aatgtttgaa gattctcaag attcttgtag aaacctggtc cctgtacaga
     1321 gagtggtgta caatgttctt agtgaactgg agaagacatt taacctgcca gttctggaag
     1381 cactgttcag cgatgtcaac atgcaggaat accccgattt aattcacatt tataaaggct
     1441 ttgaaaatgt aatccatgac aaattgcctc tccaagaaag tgaagaagaa gagagggagg
     1501 agaggtctgg cctccaacta agtcttgaac aaggaactgg tgaaaactct tttcgaagcc
     1561 tgacttggcc accttcgggt tccccatctc atgctggtac aaccccacct gaaaatggac
     1621 tctcagagca cccctgtgaa acagaacaga taaatgcaaa gagaaaagat acaaccagtg
     1681 acaaagatga ttcgctagga agccaacaaa caaatgaaca atgtgctcaa aaggctgagc
     1741 caacagagtc ctgcgaacaa attgctgtcc aagtgaataa tggggatgct ggaagggaga
     1801 tgccctgccc gttgccctgt gatgaagaaa gcccagaggc agagctacac aaccatggaa
     1861 tccaaattaa ttcctgttct gtgcgactgg tggatataaa aaaggaaaag ccattttcta
     1921 attcaaaagt tgagtgccaa gcccaagcaa gaactcatca taaccaggca tctgacataa
     1981 tagtcatcag cagtgaggac tctgaaggat ccactgacgt tgatgagccc ttagaagtct
     2041 tcatctcagc accgagaagt gagcctgtga tcaataatga caacccttta gaatcaaatg
     2101 atgaaaagga gggccaagaa gccacttgct cacgacccca gattgtacca gagcccatgg
     2161 atttcagaaa attatctaca ttcagagaaa gttttaagaa aagagtgata ggacaagacc
     2221 acgacttttc agaatccagt gaggaggagg cgcccgcaga agcctcaagc ggggcactga
     2281 gaagcaagca tggtgagaag gctcctatga cttctagaag tacatctact tggagaatac
     2341 ccagcaggaa gagacgtttc agcagtagtg acttttcaga cctgagtaat ggagaagagc
     2401 ttcaggaaac ctgcagctca tccctaagaa gagggtcagg taaagaagat tacccaactt
     2461 tcttgtacaa agtggttgat tctagaggaa agtcttaccc aactgtgagt gctgattacc
     2521 aggacgccgt tgagaaggcg aagaagaagc tcagaggctt catcgctgag aagagatgcg
     2581 ctcctctaat gctccgtttg gcattccact ctgctggaac ctttgacaag ggcacgaaga
     2641 ccggtggacc cttcggaacc atcaagcacc ctgccgaact ggctcacagc gctaacaacg
     2701 gtcttgacat cgctgttagg cttttggagc cactcaaggc ggagttccct attttgagct
     2761 acgccgattt ctaccagttg gctggcgttg ttgccgttga ggtcacgggt ggacctaagg
     2821 ttccattcca ccctggaaga gaggacaagc ctgagccacc accagagggt cgcttgcctg
     2881 atcccactaa gggttctgac catttgagag atgtgtttgg caaagctatg gggcttactg
     2941 accaagatat cgttgctcta tctgggggtc acactattgg agctgcacac aaggagcgtt
     3001 ctggatttga gggtccctgg acctctaatc ctcttatttt cgacaactca tacttcacgg
     3061 agttgttgag tggtgagaag gaaggtctcc ttcagctacc ttctgacaag gctcttttgt
     3121 ctgaccctgt attccgccct ctcgttgata aatatgcagc ggacgaagat gccttctttg
     3181 ctgattacgc tgaggctcac caaaagcttt ccgagcttgg gtttgctgat gccgactaca
     3241 aagaccatga cggtgattat aaagatcatg acatcgatta caaggatgac gatgacaagt
     3301 aactcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     3361 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3421 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3481 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     3541 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     3601 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3661 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3721 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3781 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3841 gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     3901 ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     3961 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     4021 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     4081 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     4141 acattgggga gttcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     4201 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     4261 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     4321 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     4381 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     4441 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     4501 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     4561 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     4621 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     4681 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     4741 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     4801 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     4861 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     4921 caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4981 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5041 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5101 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5161 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5221 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5281 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5341 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5401 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5461 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5521 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5581 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5641 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5701 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5761 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5821 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5881 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5941 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6001 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6061 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6121 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6181 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6241 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6301 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6361 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6421 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6481 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6541 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6601 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6661 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6721 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6781 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6841 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6901 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6961 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7021 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7081 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7141 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7201 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7261 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7321 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7381 ctgacgtc
//
