LOCUS       sequence-369149-        6662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express FLAG epitope and APEX2-tagged SRSF7 fusion protein in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6662)
  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 6662)
  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 SRSF7
FEATURES             Location/Qualifiers
     source          1..6662
                     /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(1726..1750)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1761..2507
                     /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             2508..2573
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2614..2631)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2620..2844
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2977..2996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3128..3175
                     /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             3183..4205
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4335..4456
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4372..4391)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4426..4445
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4505..4521)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4505..4521)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4518..4540)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4529..4545
                     /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(4553..4559,4560..4577,4578..4583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4598..4619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4736..4753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4907..5495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4987..5006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5666..6457,6458..6526))
                     /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     6289..6308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6527..6631)
                     /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 atgtcgcgtt
     1021 acgggcggta cggaggagaa accaaggtgt atgttggtaa cctgggaact ggcgctggca
     1081 aaggagagtt agaaagggct ttcagttatt atggtccttt aagaactgta tggattgcga
     1141 gaaatcctcc aggatttgcc tttgtggaat tcgaagatcc tagagatgca gaagatgcag
     1201 tacgaggact ggatggaaag gtgatttgtg gctcccgagt gagggttgaa ctatcgacag
     1261 gcatgcctcg gagatcacgt tttgatagac cacctgcccg acgtcccttt gatccaaatg
     1321 atagatgcta tgagtgtggc gaaaagggac attatgctta tgattgtcat cgttacagcc
     1381 ggcgaagaag aagcaggtca cggtctagat cacattctcg atccagagga aggcgatact
     1441 ctcgctcacg cagcaggagc aggggacgaa ggtcaaggtc agcatctcct cgacgatcaa
     1501 gatctatctc tcttcgtaga tcaagatcag cttcactcag aagatctagg tctggttcta
     1561 taaaaggatc gaggtatttc caatccccgt cgaggtcaag atcaagatcc aggtctattt
     1621 cacgaccaag aagcagccga tcaaagtcca gatctccatc tccaaaaaga agtcgttccc
     1681 catcaggaag tcctcgcaga agtgcaagtc ctgaaagaat ggactgccca actttcttgt
     1741 acaaagtggt tgattctaga ggaaagtctt acccaactgt gagtgctgat taccaggacg
     1801 ccgttgagaa ggcgaagaag aagctcagag gcttcatcgc tgagaagaga tgcgctcctc
     1861 taatgctccg tttggcattc cactctgctg gaacctttga caagggcacg aagaccggtg
     1921 gacccttcgg aaccatcaag caccctgccg aactggctca cagcgctaac aacggtcttg
     1981 acatcgctgt taggcttttg gagccactca aggcggagtt ccctattttg agctacgccg
     2041 atttctacca gttggctggc gttgttgccg ttgaggtcac gggtggacct aaggttccat
     2101 tccaccctgg aagagaggac aagcctgagc caccaccaga gggtcgcttg cctgatccca
     2161 ctaagggttc tgaccatttg agagatgtgt ttggcaaagc tatggggctt actgaccaag
     2221 atatcgttgc tctatctggg ggtcacacta ttggagctgc acacaaggag cgttctggat
     2281 ttgagggtcc ctggacctct aatcctctta ttttcgacaa ctcatacttc acggagttgt
     2341 tgagtggtga gaaggaaggt ctccttcagc taccttctga caaggctctt ttgtctgacc
     2401 ctgtattccg ccctctcgtt gataaatatg cagcggacga agatgccttc tttgctgatt
     2461 acgctgaggc tcaccaaaag ctttccgagc ttgggtttgc tgatgccgac tacaaagacc
     2521 atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac aagtaactcg
     2581 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     2641 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     2701 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     2761 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     2821 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     2881 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2941 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3001 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3061 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3121 gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg
     3181 ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
     3241 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
     3301 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
     3361 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
     3421 gggagttcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     3481 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
     3541 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     3601 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     3661 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     3721 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     3781 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     3841 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     3901 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     3961 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     4021 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     4081 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     4141 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     4201 aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4261 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4321 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4381 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4441 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4501 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4561 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4621 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4681 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4741 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4801 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4861 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4921 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4981 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5041 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5101 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5161 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5221 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5281 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5341 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5401 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5461 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5521 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5581 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5641 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5701 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5761 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5821 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5881 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5941 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6001 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6061 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6121 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6181 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6241 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6301 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6361 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6421 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6481 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6541 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6601 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6661 tc
//
