LOCUS       sequence-254987-        6575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express APEX2-GFP fusion in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6575)
  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 6575)
  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-GFP
FEATURES             Location/Qualifiers
     source          1..6575
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(37..625)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(117..136)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(796..1587,1588..1656))
                     /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     1419..1438
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1657..1761)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1836..1855)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2027..2406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2407..2610
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2561..2581
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2612..2630
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2633..2651
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2661..2685
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2775..3524
                     /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             3531..3557
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             join(3561..3563,3564..3566,3567..4280)
                     /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(3561..3563,3564..3566,3567..4277)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3606..3627)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3867..3886)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4214..4235
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4319..4336)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4325..4549
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4682..4701)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4833..4880
                     /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             4888..5910
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4888..5910
                     /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    6040..6161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6077..6096)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6131..6150
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6210..6226)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6210..6226)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6223..6245)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6234..6250
                     /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(6258..6264,6265..6282,6283..6288))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6303..6324
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6441..6458)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
       61 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
      121 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
      181 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
      241 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
      301 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
      361 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
      421 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
      481 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
      541 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
      601 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
      661 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
      721 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
      781 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
      841 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
      901 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
      961 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     1021 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     1081 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     1141 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     1201 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     1261 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     1321 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     1381 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     1441 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     1501 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     1561 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     1621 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     1681 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     1741 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     1801 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     1861 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     1921 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     1981 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
     2041 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     2101 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     2161 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     2221 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     2281 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     2341 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     2401 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     2461 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     2521 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     2581 gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc
     2641 agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat
     2701 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg
     2761 tttaaactta caccatggga aagtcttacc caactgtgag tgctgattac caggacgccg
     2821 ttgagaaggc gaagaagaag ctcagaggct tcatcgctga gaagagatgc gctcctctaa
     2881 tgctccgttt ggcattccac tctgctggaa cctttgacaa gggcacgaag accggtggac
     2941 ccttcggaac catcaagcac cctgccgaac tggctcacag cgctaacaac ggtcttgaca
     3001 tcgctgttag gcttttggag ccactcaagg cggagttccc tattttgagc tacgccgatt
     3061 tctaccagtt ggctggcgtt gttgccgttg aggtcacggg tggacctaag gttccattcc
     3121 accctggaag agaggacaag cctgagccac caccagaggg tcgcttgccc gatcccacta
     3181 agggttctga ccatttgaga gatgtgtttg gcaaagctat ggggcttact gaccaagata
     3241 tcgttgctct atctgggggt cacactattg gagctgcaca caaggagcgt tctggatttg
     3301 agggtccctg gacctctaat cctcttattt tcgacaactc atacttcacg gagttgttga
     3361 gtggtgagaa ggaaggtctc cttcagctac cttctgacaa ggctcttttg tctgaccctg
     3421 tattccgccc tctcgttgac aaatatgcag cggacgaaga tgccttcttt gctgattacg
     3481 ctgaggctca ccaaaagctt tccgagcttg ggtttgctga tgccctgcag ctgcctcccc
     3541 tggagcgcct gaccctggac atggtgagca agggcgagga gctgttcacc ggggtggtgc
     3601 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     3661 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     3721 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     3781 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     3841 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     3901 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     3961 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     4021 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     4081 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     4141 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     4201 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     4261 tggacgagct gtacaagtga gctcgagtct agagggcccg tttaaacccg ctgatcagcc
     4321 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4381 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     4441 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     4501 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     4561 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     4621 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     4681 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4741 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4801 aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct
     4861 ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag
     4921 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     4981 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     5041 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     5101 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     5161 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     5221 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     5281 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     5341 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     5401 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     5461 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     5521 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     5581 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     5641 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     5701 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     5761 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     5821 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     5881 cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc
     5941 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     6001 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     6061 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     6121 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     6181 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     6241 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     6301 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6361 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6421 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     6481 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     6541 gcaggaaaga acatgtgagc aaaaggccag caaaa
//
