LOCUS       sequence-254975-        6509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express APEX2 fusion to cap-binding protein eIF4E1 in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6509)
  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 6509)
  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 APEX2-eIF4E1
FEATURES             Location/Qualifiers
     source          1..6509
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(172..963,964..1032))
                     /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     795..814
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1033..1137)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1212..1231)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1403..1782
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1783..1986
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1937..1957
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1988..2006
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2009..2027
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2037..2061
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2151..2900
                     /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="MGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAG
                     TFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVA
                     VEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGH
                     TIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVD
                     KYAADEDAFFADYAEAHQKLSELGFADA"
     CDS             2907..2933
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(3629..3646)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3635..3859
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3992..4011)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4143..4190
                     /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             4198..5220
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4198..5220
                     /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    5350..5471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5387..5406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5441..5460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5520..5536)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5520..5536)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5533..5555)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5544..5560
                     /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(5568..5574,5575..5592,5593..5598))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5613..5634
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5751..5768)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5922..6509,1))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6002..6021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
       61 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
      121 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
      181 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
      241 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
      301 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
      361 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
      421 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
      481 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
      541 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
      601 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
      661 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
      721 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
      781 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
      841 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
      901 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
      961 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     1021 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     1081 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     1141 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     1201 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     1261 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     1321 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     1381 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     1441 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     1501 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     1561 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     1621 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     1681 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     1741 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     1801 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     1861 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     1921 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     1981 agagctctcc ctatcagtga tagagatctc cctatcagtg atagagatcg tcgacgagct
     2041 cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga
     2101 agacaccggg accgatccag cctccggact ctagcgttta aacttacacc atgggaaagt
     2161 cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag aagaagctca
     2221 gaggcttcat cgctgagaag agatgcgctc ctctaatgct ccgtttggca ttccactctg
     2281 ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc aagcaccctg
     2341 ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt ttggagccac
     2401 tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct ggcgttgttg
     2461 ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag gacaagcctg
     2521 agccaccacc agagggtcgc ttgcccgatc ccactaaggg ttctgaccat ttgagagatg
     2581 tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct gggggtcaca
     2641 ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc tctaatcctc
     2701 ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa ggtctccttc
     2761 agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc gttgacaaat
     2821 atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa aagctttccg
     2881 agcttgggtt tgctgatgcc ctgcagctgc ctcccctgga gcgcctgacc ctggacatgg
     2941 cgactgtcga accggaaacc acccctactc ctaatccccc gactacagaa gaggagaaaa
     3001 cggaatctaa tcaggaggtt gctaacccag aacactatat taaacatccc ctacagaaca
     3061 gatgggcact ctggtttttt aaaaatgata aaagcaaaac ttggcaagca aacctgcggc
     3121 tgatctccaa gtttgatact gttgaagact tttgggctct gtacaaccat atccagttgt
     3181 ctagtaattt aatgcctggc tgtgactact cactttttaa ggatggtatt gagcctatgt
     3241 gggaagatga gaaaaacaaa cggggaggac gatggctaat tacattgaac aaacagcaga
     3301 gacgaagtga cctcgatcgc ttttggctag agacacttct gtgccttatt ggagaatctt
     3361 ttgatgacta cagtgatgat gtatgtggcg ctgttgttaa tgttagagct aaaggtgata
     3421 agatagcaat atggactact gaatgtgaaa acagagaagc tgttacacat atagggaggg
     3481 tatacaagga aaggttagga cttcctccaa agatagtgat tggttatcag tcccacgcag
     3541 acacagctac taagagcggc tccaccacta aaaataggtt tgttgtttaa gctcgagtct
     3601 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     3661 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     3721 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     3781 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     3841 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     3901 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3961 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4021 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4081 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc
     4141 tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc cttggccaaa
     4201 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     4261 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     4321 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     4381 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     4441 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     4501 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     4561 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     4621 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     4681 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     4741 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     4801 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     4861 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     4921 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     4981 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     5041 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     5101 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     5161 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     5221 cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     5281 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5341 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5401 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5461 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5521 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5581 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5641 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5701 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5761 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5821 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5881 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5941 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6001 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6061 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6121 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6181 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6241 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6301 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6361 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6421 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6481 cagattacgc gcagaaaaaa aggatctca
//
