LOCUS       sequence-367864-        7591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express FLAG epitope and APEX2-tagged WRAP53 fusion protein in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7591)
  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 7591)
  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 WRAP53
FEATURES             Location/Qualifiers
     source          1..7591
                     /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"
     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(3477..3501)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3543..3560)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3549..3773
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3906..3925)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4057..4104
                     /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             4112..5134
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5264..5385
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5301..5320)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5355..5374
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5434..5450)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5434..5450)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5447..5469)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5458..5474
                     /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(5482..5488,5489..5506,5507..5512))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5527..5548
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5665..5682)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5836..6424)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5916..5935)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6595..7386,7387..7455))
                     /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     7218..7237
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7456..7560)
                     /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 aagcaggctt caagactttg gagactcaac cgttagctcc
     1861 ggactgctgt ccttcagacc aggacccagc tccagcccat ccttctcccc acgcttcccc
     1921 gatgaataaa aatgcggact ctgaactgat gccaccgcct cccgaaaggg gggatccgcc
     1981 ccggttgtcc ccagatcctg tggctggctc agctgtgtcc caggagctac gggaggggga
     2041 cccagtttct ctctccactc ccctggaaac agagtttggt tcccctagtg agttgagtcc
     2101 tcgaatcgag gagcaagaac tttctgaaaa tacaagcctt cctgcagaag aagcaaacgg
     2161 gagcctttct gaagaagaag cgaacgggcc agagttgggg tctggaaaag ccatggaaga
     2221 tacctctggg gaacccgctg cagaggacga gggagacacc gcttggaact acagcttctc
     2281 ccagctgcct cgatttctca gtggttcctg gtcagagttc agcacccaac ctgagaactt
     2341 cttgaaaggc tgtaagtggg ctcctgacgg ttcctgcatc ttgaccaata gtgctgataa
     2401 catcttgcga atttataacc tgcccccaga gctgtaccat gagggggagc aggtggaata
     2461 tgcagaaatg gtccctgtcc ttcgaatggt ggaaggtgat accatctatg attactgctg
     2521 gtattctctg atgtcctcag cccagccaga cacctcctac gtggccagca gcagccggga
     2581 gaacccgatt catatctggg acgcattcac tggagagctc cgggcttcct ttcgcgccta
     2641 caaccacctg gatgagctga cggcagccca ttcgctctgc ttctccccgg atggctccca
     2701 gctcttctgt ggcttcaacc ggactgtgcg tgttttttcc acggcccggc ctggccgaga
     2761 ctgcgaggtc cgagccacat ttgcaaaaaa gcagggccag agcggcatca tctcctgcat
     2821 agccttcagc ccagcccagc ccctctatgc ctgtggctcc tacggccgct ccctgggtct
     2881 gtatgcctgg gatgatggct cccctctcgc cttgctggga gggcaccaag ggggcatcac
     2941 ccacctctgc tttcatcccg atggcaaccg cttcttctca ggagcccgca aggatgctga
     3001 gctcctgtgc tgggatctcc ggcagtctgg ttacccactg tggtccctgg gtcgagaggt
     3061 gaccaccaat cagcgcatct acttcgatct ggacccgacc gggcagttcc tagtgagtgg
     3121 cagcacgagc ggggctgtct ctgtgtggga cacggacggg cctggcaatg atgggaagcc
     3181 ggagcccgtg ttgagttttc tgccccagaa ggactgcacc aatggcgtga gcctgcaccc
     3241 tagcctgcct ctcctggcca ctgcctccgg tcagcgtgtg tttcctgagc ccacagagag
     3301 tggggacgaa ggagaggagc tgggccttcc cttgctctcc acgcgccacg tccaccttga
     3361 atgtcggctt cagctctggt ggtgtggggg ggcgccagac tccagcatcc ctgatgatca
     3421 ccagggcgag aaagggcagg gaggaacgga gggaggtgtg ggtgagctga tataaaaccc
     3481 agctttcttg tacaaagtgg tctgactcga gtctagaggg cccgtttaaa cccgctgatc
     3541 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3601 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3661 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3721 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3781 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3841 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3901 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3961 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4021 caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta
     4081 ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt
     4141 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     4201 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     4261 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     4321 gctcccgatt ccggaagtgc ttgacattgg ggagttcagc gagagcctga cctattgcat
     4381 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     4441 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     4501 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     4561 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     4621 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     4681 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     4741 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     4801 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     4861 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     4921 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     4981 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     5041 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     5101 acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac
     5161 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     5221 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     5281 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     5341 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     5401 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5461 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5521 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5581 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5641 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5701 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5761 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5821 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5881 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5941 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6001 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6061 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6121 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6181 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6241 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6301 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6361 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6421 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6481 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6541 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6601 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6661 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6721 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6781 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6841 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6901 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6961 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     7021 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7081 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7141 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7201 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7261 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7321 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7381 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7441 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7501 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7561 cacatttccc cgaaaagtgc cacctgacgt c
//
