LOCUS       sequence-360428-        7599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express FLAG epitope and APEX2-tagged fusion protein in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7599)
  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 7599)
  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: pDEST-cDNA5-FRT/TO-3*Flag-APEX2 N-term
FEATURES             Location/Qualifiers
     source          1..7599
                     /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..1929
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1954..1959,1960..1977,1978..1984)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2038..2697
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2104..2123)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3039..3344
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMLTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3385..3509)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3551..3568)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3557..3781
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3914..3933)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4065..4112
                     /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             4120..5142
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5272..5393
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5309..5328)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5363..5382
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5442..5458)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5442..5458)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5455..5477)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5466..5482
                     /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(5490..5496,5497..5514,5515..5520))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5535..5556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5673..5690)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5844..6432)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5924..5943)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6603..7394,7395..7463))
                     /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     7226..7245
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7464..7568)
                     /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 aagctgaacg agaaacgtaa aatgatataa atatcaatat
     1861 attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca
     1921 gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     1981 aatgtgtgga ttttgagtta ggatccgtcg agattttcag gagctaagga agctaaaatg
     2041 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
     2101 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
     2161 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     2221 attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag
     2281 ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg
     2341 ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca catatattcg
     2401 caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat
     2461 atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc
     2521 aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac
     2581 aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc
     2641 ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaaaga
     2701 tctggatccg gcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc
     2761 ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg tatgctatga
     2821 agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga
     2881 tgtcaatatc tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc
     2941 cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat
     3001 gaacggctct tttgctgacg agaacagggg ctggtgaaat gcagtttaag gtttacacct
     3061 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc
     3121 ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc
     3181 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgttg accaccgata
     3241 tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa
     3301 atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta
     3361 tacacagcca gtctgcaggt cgaccatagt gactggatat gttgtgtttt acagtattat
     3421 gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt
     3481 tctcgttcag ctttcttgta caaagtggtc tgactcgagt ctagagggcc cgtttaaacc
     3541 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3601 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3661 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3721 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3781 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3841 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3901 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3961 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4021 ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga
     4081 agttcctatt ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg
     4141 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     4201 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     4261 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     4321 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg agttcagcga gagcctgacc
     4381 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     4441 cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
     4501 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     4561 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
     4621 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
     4681 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
     4741 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
     4801 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
     4861 ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc
     4921 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
     4981 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
     5041 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
     5101 ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc
     5161 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     5221 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     5281 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5341 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5401 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5461 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5521 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5581 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5641 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5701 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5761 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5821 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5881 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5941 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6001 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6061 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6121 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6181 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6241 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6301 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6361 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6421 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6481 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6541 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6601 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6661 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6721 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6781 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6841 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6901 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6961 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7021 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7081 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7141 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7201 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7261 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7321 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7381 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7441 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7501 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7561 gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
