LOCUS       sequence-367892-        7519 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express FLAG epitope and APEX2-tagged PSPC1 fusion protein in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7519)
  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 7519)
  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 PSPC1
FEATURES             Location/Qualifiers
     source          1..7519
                     /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"
     CDS             3183..3194
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     protein_bind    complement(3405..3429)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3471..3488)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3477..3701
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3834..3853)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3985..4032
                     /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             4040..5062
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5192..5313
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5229..5248)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5283..5302
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5362..5378)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5362..5378)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5375..5397)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5386..5402
                     /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(5410..5416,5417..5434,5435..5440))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5455..5476
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5593..5610)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5764..6352)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5844..5863)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6523..7314,7315..7383))
                     /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     7146..7165
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7384..7488)
                     /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 aagttggcac catgatgtta agaggaaacc tgaagcaagt
     1861 gcgcattgag aaaaacccgg cccgccttcg cgccctggag tccgcggtgg gcgagagcga
     1921 gccggcggcc gcggcagcca tggcgctcgc tcttgccggg gagccggcac cgcccgcgcc
     1981 cgcgcctcca gaggaccacc cggacgagga gatggggttc actatcgaca tcaagagttt
     2041 cctcaagccg ggcgagaaga cgtacacgca gcgctgccgc ctcttcgtgg gaaatctgcc
     2101 caccgacatc acggaggagg acttcaagag gctcttcgaa cgctatggcg agcccagcga
     2161 agtcttcatc aaccgggacc gtggcttcgg cttcatccgc ttggaatcca gaaccctggc
     2221 tgaaattgca aaagcagagc tggacggcac cattctcaag agcagacctc tacggattcg
     2281 cttcgctaca catggagcag ccttgactgt caagaacctt tctccagttg tttccaatga
     2341 gctgctagag caagcatttt ctcagtttgg tccagtagag aaagctgttg tggttgtgga
     2401 tgatcgcggt agagctacag gaaaaggttt tgtagagttt gcagcaaaac ctcctgcacg
     2461 aaaggctctg gaaagatgtg gtgatggggc attcttgcta acaacgaccc ctcgtccagt
     2521 cattgtggaa cccatggagc agtttgatga tgaagatggc ttgccagaga agctgatgca
     2581 gaaaactcaa caatatcata aggaaagaga acaaccacca cgttttgctc aacctgggac
     2641 atttgaattt gagtatgcat ctcgatggaa ggctcttgat gaaatggaaa agcagcagcg
     2701 tgagcaggtt gatagaaaca tcagagaagc caaagagaaa ctggaggcag aaatggaagc
     2761 agctaggcat gaacaccaat taatgctaat gaggcaagat ctaatgaggc gtcaagaaga
     2821 actcagacgc ttggaagaac tcagaaacca agagttgcaa aaacggaagc aaatacaact
     2881 aagacatgaa gaggagcatc ggcggcgtga ggaagaaatg atccgacaca gagaacagga
     2941 ggaactgagg cgacagcaag agggctttaa gccaaactac atggaaaata gagaacagga
     3001 aatgagaatg ggtgatatgg gtccccgtgg agcaataaac atgggagatg cgtttagccc
     3061 agcccctgct ggtaaccaag gtcctcctcc aatgatgggt atgaatatga acaacagagc
     3121 aactatacct ggcccaccaa tgggtcctgg tcctgccatg ggaccagaag gagccgcaaa
     3181 tatgggaact ccaatgatgc cagataatgg agcagtgcac aatgacagat ttcctcaagg
     3241 accaccatct cagatgggtt cacctatggg gagtagaaca ggttctgaaa cccctcaagc
     3301 accaatgagt ggtgtaggtc ctgtgagtgg tggtcctggt ggctttggta gaggaagtca
     3361 agggggcaac tttgaaggcc ctaataagcg tcgtagatat tgatacccaa ctttcttgta
     3421 caaagtggtc tgactcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     3481 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3541 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3601 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3661 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3721 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3781 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3841 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3901 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3961 ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag
     4021 tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     4081 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
     4141 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     4201 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     4261 ggaagtgctt gacattgggg agttcagcga gagcctgacc tattgcatct cccgccgtgc
     4321 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     4381 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     4441 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     4501 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     4561 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     4621 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     4681 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     4741 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     4801 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     4861 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     4921 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     4981 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     5041 tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta
     5101 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     5161 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     5221 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     5281 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     5341 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5401 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5461 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5521 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5581 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5641 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5701 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5761 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5821 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5881 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5941 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6001 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6061 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6121 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6181 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6241 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6301 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6361 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6421 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6481 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6541 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6601 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6661 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6721 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6781 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6841 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6901 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6961 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7021 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7081 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7141 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7201 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7261 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7321 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7381 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7441 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7501 aaaagtgcca cctgacgtc
//
