LOCUS       sequence-367945-        8291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express FLAG epitope and APEX2-tagged PML fusion protein in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8291)
  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 8291)
  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 PML
FEATURES             Location/Qualifiers
     source          1..8291
                     /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(3355..3379)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3390..4136
                     /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             4137..4202
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4243..4260)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4249..4473
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4606..4625)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4757..4804
                     /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             4812..5834
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5964..6085
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6001..6020)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6055..6074
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6134..6150)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6134..6150)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6147..6169)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6158..6174
                     /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(6182..6188,6189..6206,6207..6212))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6227..6248
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6365..6382)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6536..7124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6616..6635)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7295..8086,8087..8155))
                     /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     7918..7937
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8156..8260)
                     /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 atggagcctg
     1021 cacccgcccg atctccgagg ccccagcagg accccgcccg gccccaggag cccaccatgc
     1081 ctccccccga gaccccctct gaaggccgcc agcccagccc cagccccagc cctacagagc
     1141 gagcccccgc ttcggaggag gagttccagt ttctgcgctg ccagcaatgc caggcggaag
     1201 ccaagtgccc gaagctgctg ccttgtctgc acacgctgtg ctcaggatgc ctggaggcgt
     1261 cgggcatgca gtgccccatc tgccaggcgc cctggcccct aggtgcagac acacccgccc
     1321 tggataacgt ctttttcgag agtctgcagc ggcgcctgtc ggtgtaccgg cagattgtgg
     1381 atgcgcaggc tgtgtgcacc cgctgcaaag agtcggccga cttctggtgc tttgagtgcg
     1441 agcagctcct ctgcgccaag tgcttcgagg cacaccagtg gttcctcaag cacgaggccc
     1501 ggcccctagc agagctgcgc aaccagtcgg tgcgtgagtt cctggacggc acccgcaaga
     1561 ccaacaacat cttctgctcc aaccccaacc accgcacccc tacgctgacc agcatctact
     1621 gccgaggatg ttccaagccg ctgtgctgct cgtgcgcgct ccttgacagc agccacagtg
     1681 atctcaagtg cgacatcagc gcagagatcc agcagcgaca ggaggagctg gacgccatga
     1741 cgcaggcgct gcaggagcag gatagtgcct ttggcgcggt tcacgcgcag atgcacgcgg
     1801 ctgtcggcca gctgggccgc gcgcgtgccg agaccgagga gctgatccgc gagcgcgtgc
     1861 gccaggtggt agctcacgtg cgggctcagg agcgcgagct gctggaggct gtggacgcgc
     1921 ggtaccagcg cgactacgag gagatggcca gtcggctggg ccgcctggat gctgtgctgc
     1981 agcgcatccg cacgggcagc gcgctggtgc agaggatgaa gtgctacgcc tcggaccagg
     2041 aggtgctgga catgcacggt ttcctgcgcc aggcgctctg ccgcctgcgc caggaggagc
     2101 cccagagcct gcaagctgcc gtgcgcaccg atggcttcga cgagttcaag gtgcgcctgc
     2161 aggacctcag ctcttgcatc acccagggga aagatgcagc tgtatccaag aaagccagcc
     2221 cagaggctgc cagcactccc agggacccta ttgacgttga cctggatgtc tccaatacaa
     2281 cgacagccca gaagaggaag tgcagccaga cccagtgccc caggaaggtc atcaagatgg
     2341 agtctgagga ggggaaggag gcaaggttgg ctcggagctc cccggagcag cccaggccca
     2401 gcacctccaa ggcagtctca ccaccccacc tggatggacc gcctagcccc aggagccccg
     2461 tcataggaag tgaggtcttc ctgcccaaca gcaaccacgt ggccagtggc gccggggagg
     2521 cagaggaacg cgttgtggtg atcagcagct cggaagactc agatgccgaa aactcgtgca
     2581 tggagcccat ggagaccgcc gagccacagt cctcgccagc ccactcctcg ccagcccact
     2641 cctcgccagc ccactcctcg ccagtccagt ctctgctgag agcacaagga gcctccagcc
     2701 tgccctgtgg cacataccac cccccagctt ggcctcccca ccagcccgct gagcaggctg
     2761 ccacccccga tgctgagcct cacagcgagc ctcctgatca ccaggagcgc cctgccgtcc
     2821 accgtgggat ccgctacctg ttgtacagag cacagagagc catccgcctt cgccatgccc
     2881 tccgcttgca ccctcaattg catcgggccc ctattcggac ttggtctccc catgtggtcc
     2941 aagccagcac tcctgccatc acagggcccc tcaaccatcc tgccaatgcc caggaacatc
     3001 ctgcccagct gcaaaggggc atcagcccac cccaccggat acgaggggct gtgcgatccc
     3061 gcagccgctc cctccggggc tcctcccatt tatcccagtg gctcaacaac ttttttgccc
     3121 tccccttctc ctccatggct tcccagcttg acatgtcttc cgtggtgggg gcaggggaag
     3181 gcagagccca gactcttgga gcagttgttc cccctgggga ctctgtcaga ggctccatgg
     3241 aggcctctca agtccaagtg cctctggaag cctctccaat tacattccca ccaccctgtg
     3301 ccccagaaag gccccccatc agcccagtcc caggcgcccg tcaagcaggc ctctgcccaa
     3361 ctttcttgta caaagtggtt gattctagag gaaagtctta cccaactgtg agtgctgatt
     3421 accaggacgc cgttgagaag gcgaagaaga agctcagagg cttcatcgct gagaagagat
     3481 gcgctcctct aatgctccgt ttggcattcc actctgctgg aacctttgac aagggcacga
     3541 agaccggtgg acccttcgga accatcaagc accctgccga actggctcac agcgctaaca
     3601 acggtcttga catcgctgtt aggcttttgg agccactcaa ggcggagttc cctattttga
     3661 gctacgccga tttctaccag ttggctggcg ttgttgccgt tgaggtcacg ggtggaccta
     3721 aggttccatt ccaccctgga agagaggaca agcctgagcc accaccagag ggtcgcttgc
     3781 ctgatcccac taagggttct gaccatttga gagatgtgtt tggcaaagct atggggctta
     3841 ctgaccaaga tatcgttgct ctatctgggg gtcacactat tggagctgca cacaaggagc
     3901 gttctggatt tgagggtccc tggacctcta atcctcttat tttcgacaac tcatacttca
     3961 cggagttgtt gagtggtgag aaggaaggtc tccttcagct accttctgac aaggctcttt
     4021 tgtctgaccc tgtattccgc cctctcgttg ataaatatgc agcggacgaa gatgccttct
     4081 ttgctgatta cgctgaggct caccaaaagc tttccgagct tgggtttgct gatgccgact
     4141 acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat gacgatgaca
     4201 agtaactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     4261 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     4321 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     4381 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     4441 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     4501 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4561 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4621 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4681 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     4741 atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa
     4801 cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
     4861 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
     4921 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
     4981 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
     5041 ttgacattgg ggagttcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
     5101 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
     5161 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
     5221 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
     5281 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
     5341 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     5401 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     5461 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     5521 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     5581 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     5641 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
     5701 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
     5761 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
     5821 gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     5881 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5941 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     6001 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     6061 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     6121 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     6181 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     6241 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6301 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     6361 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6421 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6481 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6541 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6601 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6661 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6721 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6781 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6841 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6901 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6961 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     7021 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     7081 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     7141 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     7201 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7261 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7321 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7381 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7441 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7501 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7561 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7621 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7681 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7741 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7801 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7861 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7921 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7981 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     8041 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     8101 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     8161 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8221 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     8281 cacctgacgt c
//
