LOCUS       sequence-254965-        6659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express ER-localized C1-APEX2 fusion in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6659)
  AUTHORS   Padron A, Iwasaki S, Ingolia NT
  TITLE     Proximity RNA Labeling by APEX-Seq Reveals the Organization of 
            Translation Initiation Complexes and Repressive RNA Granules.
  JOURNAL   Mol Cell. 2019 Aug 22;75(4):875-887.e5. doi: 
            10.1016/j.molcel.2019.07.030.
  PUBMED    31442426
REFERENCE   2  (bases 1 to 6659)
  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.2019.07"; journalName: "Mol Cell"; date: 
            "2019-08-22- 22"; volume: "75"; issue: "4"; pages: "875-887.e5"
COMMENT     Sequence Label: pcDNA5/FRT/TO C1-APEX2
FEATURES             Location/Qualifiers
     source          1..6659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    589..607
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    610..628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     638..662
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             839..1585
                     /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             1592..1618
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             join(1622..1624,1625..1627,1628..2341)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1622..1624,1625..1627,1628..2338)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1667..1688)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1928..1947)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2275..2296
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2380..2397)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2386..2610
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2743..2762)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2894..2941
                     /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             2949..3971
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2949..3971
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4101..4222
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4138..4157)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4192..4211
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4271..4287)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4271..4287)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4284..4306)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4295..4311
                     /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(4319..4325,4326..4343,4344..4349))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4364..4385
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4502..4519)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4673..5261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4753..4772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5432..6223,6224..6292))
                     /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     6055..6074
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6293..6397)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6472..6491)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
      601 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
      661 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      721 gcctccggac tctagcgttt aaacttacac catggacccc gtggttgtac ttgggttgtg
      781 cttgtcttgc cttctgctgc tctctttgtg gaagcaaagt tacggggggg gaaagctggg
      841 aaagtcttac ccaactgtga gtgctgatta ccaggacgcc gttgagaagg cgaagaagaa
      901 gctcagaggc ttcatcgctg agaagagatg cgctcctcta atgctccgtt tggcattcca
      961 ctctgctgga acctttgaca agggcacgaa gaccggtgga cccttcggaa ccatcaagca
     1021 ccctgccgaa ctggctcaca gcgctaacaa cggtcttgac atcgctgtta ggcttttgga
     1081 gccactcaag gcggagttcc ctattttgag ctacgccgat ttctaccagt tggctggcgt
     1141 tgttgccgtt gaggtcacgg gtggacctaa ggttccattc caccctggaa gagaggacaa
     1201 gcctgagcca ccaccagagg gtcgcttgcc cgatcccact aagggttctg accatttgag
     1261 agatgtgttt ggcaaagcta tggggcttac tgaccaagat atcgttgctc tatctggggg
     1321 tcacactatt ggagctgcac acaaggagcg ttctggattt gagggtccct ggacctctaa
     1381 tcctcttatt ttcgacaact catacttcac ggagttgttg agtggtgaga aggaaggtct
     1441 ccttcagcta ccttctgaca aggctctttt gtctgaccct gtattccgcc ctctcgttga
     1501 caaatatgca gcggacgaag atgccttctt tgctgattac gctgaggctc accaaaagct
     1561 ttccgagctt gggtttgctg atgccctgca gctgcctccc ctggagcgcc tgaccctgga
     1621 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1681 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1741 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1801 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1861 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1921 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1981 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2041 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     2101 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     2161 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2221 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2281 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtg
     2341 agctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2401 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2461 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2521 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2581 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2641 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2701 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2761 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2821 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2881 gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     2941 ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     3001 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     3061 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     3121 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     3181 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     3241 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     3301 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     3361 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     3421 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     3481 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     3541 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     3601 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     3661 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     3721 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     3781 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     3841 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     3901 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     3961 caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4021 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4081 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4141 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4201 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4261 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4321 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4381 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4441 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4501 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4561 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4621 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4681 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4741 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4801 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4861 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4921 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4981 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5041 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5101 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5161 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5221 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5281 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5341 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5401 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5461 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5521 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5581 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5641 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5701 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5761 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5821 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5881 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5941 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6001 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6061 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6121 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6181 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6241 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6301 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6361 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6421 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     6481 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     6541 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     6601 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgc
//
