LOCUS       sequence-367850-        6862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express FLAG epitope and APEX2-tagged RNPS1 fusion protein in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6862)
  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 6862)
  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 RNPS1
FEATURES             Location/Qualifiers
     source          1..6862
                     /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(2748..2772)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2814..2831)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2820..3044
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3177..3196)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3328..3375
                     /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             3383..4405
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4535..4656
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4572..4591)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4626..4645
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4705..4721)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4705..4721)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4718..4740)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4729..4745
                     /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(4753..4759,4760..4777,4778..4783))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4798..4819
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4936..4953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5107..5695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5187..5206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5866..6657,6658..6726))
                     /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     6489..6508
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6727..6831)
                     /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 cgatttatca ggagtgaaaa agaagagctt
     1861 gctaggagtc aaagaaaata ataaaaagtc cagcactagg gctccttcac ctaccaaacg
     1921 caaagaccgc tcagatgaga agtccaagga tcgctcaaaa gataaagggg ccaccaagga
     1981 gtcgagtgag aaggatcgcg gccgggacaa aacccgaaag aggcgcagcg cttccagtgg
     2041 tagcagcagt accaggtctc ggtccagctc gacttccagc tcaggctcca gcaccagcac
     2101 tggctcaagc agtggctcca gctcttcctc agcatccagc cgctcaggaa gctccagcac
     2161 ctcccgcagc tccagctcta gcagctcttc tggctctcca agtccttctc ggcgcagaca
     2221 cgacaacagg aggcgctccc gctccaaatc caaaccacct aaaagagatg aaaaggagag
     2281 gaaaaggcgg agcccatctc ctaagcccac caaagtgcac attgggagac tcacccggaa
     2341 tgtgacaaag gatcacatca tggagatatt ttccacctat gggaaaatta aaatgattga
     2401 catgcccgtg gaaaggatgc atccccatct gtccaaaggc tatgcgtacg tagagtttga
     2461 gaatccagat gaagccgaga aggcgctgaa gcacatggat ggaggacaaa ttgatggcca
     2521 ggagatcact gccaccgccg tgctggcccc ctggcctagg ccacccccca ggagattcag
     2581 ccctcccagg agaatgttgc caccaccgcc tatgtggcgc aggtctcccc cacggatgag
     2641 gagaaggtcc cgctccccga ggcgcaggtc ccccgtgcgc cggagatcac ggtccccggg
     2701 ccgccgccgc cacaggagcc gctccagctc caactcctcc cgataaaacc cagctttctt
     2761 gtacaaagtg gtctgactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     2821 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2881 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2941 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3001 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3061 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3121 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3181 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3241 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3301 tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga
     3361 aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt
     3421 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
     3481 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
     3541 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat
     3601 tccggaagtg cttgacattg gggagttcag cgagagcctg acctattgca tctcccgccg
     3661 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc
     3721 ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
     3781 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
     3841 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
     3901 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
     3961 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
     4021 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
     4081 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
     4141 ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact
     4201 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
     4261 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
     4321 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
     4381 cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt
     4441 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4501 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4561 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4621 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     4681 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4741 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4801 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4861 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4921 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4981 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5041 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5101 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5161 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5221 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5281 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5341 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5401 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5461 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5521 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5581 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5641 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5701 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5761 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5821 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5881 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5941 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6001 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6061 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6121 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6181 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6241 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6301 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6361 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6421 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6481 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6541 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6601 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6661 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6721 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6781 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6841 ccgaaaagtg ccacctgacg tc
//
