LOCUS       sequence-342354-        5716 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for expression of LifeAct APEX2 in mammalian cells. Parton 
            lab clone FTE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5716)
  AUTHORS   Rae J, Ferguson C, Ariotti N, Webb RI, Cheng HH, Mead JL, Riches JD,
            Hunter DJ, Martel N, Baltos J, Christopoulos A, Bryce NS, Cagigas 
            ML, Fonseka S, Sayre ME, Hardeman EC, Gunning PW, Gambin Y, Hall TE,
            Parton RG
  TITLE     A robust method for particulate detection of a genetic tag for 3D 
            electron microscopy.
  JOURNAL   Elife. 2021 Apr 27;10. pii: 64630. doi: 10.7554/eLife.64630.
  PUBMED    33904409
REFERENCE   2  (bases 1 to 5716)
  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"; journalName: "Elife."; 
            date: "2021-04-27"; volume: "10. pii: 64630. doi"; pages: " 
            10.7554/eLife.64630"
COMMENT     Sequence Label: LifeAct-EGFP-APEX2 in pCSDEST2
FEATURES             Location/Qualifiers
     source          1..5716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    108..132
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      133..142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             139..189
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     regulatory      205..214
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(211..213,214..216,217..927)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(211..213,214..216,217..927)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(256..277)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(517..536)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     864..885
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(932..956)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             958..1704
                     /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    complement(1708..1728)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    1768..1902
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1792..1811)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1846..1865
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2015..2035)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2015..2033)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2054..2070)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2054..2070)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2067..2089)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2078..2094
                     /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(2102..2108,2109..2126,2127..2132))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2147..2168
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2285..2302)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2456..3044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2536..2555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3215..4006,4007..4075))
                     /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     3838..3857
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4076..4180)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4206..4661)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4343..4364)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4555..4574
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4732..5716
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca aggatcaaca agtttgtaca
      121 aaaaagcagg ctgccaccat gggcgtggcc gacctgatca agaagttcga gagcatcagc
      181 aaggaggagg gcgacccccc cgtggccacc atggtgagca agggcgagga gctgttcacc
      241 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      301 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      361 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
      421 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      481 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      541 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
      601 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
      661 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
      721 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
      781 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
      841 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
      901 actctcggca tggacgagct gtacaagggg tacccagctt tcttgtacaa agtggctgga
      961 aagtcttacc caactgtgag tgctgattac caggacgccg ttgagaaggc gaagaagaag
     1021 ctcagaggct tcatcgctga gaagagatgc gctcctctaa tgctccgttt ggcattccac
     1081 tctgctggaa cctttgacaa gggcacgaag accggtggac ccttcggaac catcaagcac
     1141 cctgccgaac tggctcacag cgctaacaac ggtcttgaca tcgctgttag gcttttggag
     1201 ccactcaagg cggagttccc tattttgagc tacgccgatt tctaccagtt ggctggcgtt
     1261 gttgccgttg aggtcacggg tggacctaag gttccattcc accctggaag agaggacaag
     1321 cctgagccac caccagaggg tcgcttgccc gatcccacta agggttctga ccatttgaga
     1381 gatgtgtttg gcaaagctat ggggcttact gaccaagata tcgttgctct atctgggggt
     1441 cacactattg gagctgcaca caaggagcgt tctggatttg agggtccctg gacctctaat
     1501 cctcttattt tcgacaactc atacttcacg gagttgttga gtggtgagaa ggaaggtctc
     1561 cttcagctac cttctgacaa ggctcttttg tctgaccctg tattccgccc tctcgttgac
     1621 aaatatgcag cggacgaaga tgccttcttt gctgattacg ctgaggctca ccaaaagctt
     1681 tccgagcttg ggtttgctga tgcctaacaa ctttattata catagttgca ctcgagcctc
     1741 tagaactata gtgagtcgta ttacgtagat ccagacatga taagatacat tgatgagttt
     1801 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     1861 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     1921 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaattcgc ggccgcggcg
     1981 ccaatgcatt gggcccggta cccagctttt gttcccttta gtgagggtta attgcgcgct
     2041 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2101 acaacatacg agccgggagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2161 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2221 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2281 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2341 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2401 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2461 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2521 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2581 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2641 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2701 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2761 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2821 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     2881 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     2941 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3001 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3061 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3121 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3181 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3241 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3301 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3361 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3421 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3481 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3541 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3601 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3661 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3721 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     3781 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     3841 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     3901 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     3961 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4021 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4081 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4141 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4201 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     4261 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     4321 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     4381 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     4441 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     4501 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     4561 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     4621 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
     4681 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agtcgaccgc
     4741 caattcaata tggcgtatat ggactcatgc caattcaata tggtggatct ggacctgtgc
     4801 caattcaata tggcgtatat ggactcgtgc caattcaata tggtggatct ggaccccagc
     4861 caattcaata tggcggactt ggcaccatgc caattcaata tggcggacct ggcactgtgc
     4921 caactgggga ggggtctact tggcacggtg ccaagtttga ggaggggtct tggccctgtg
     4981 ccaagtccgc catattgaat tggcatggtg ccaataatgg cggccatatt ggctatatgc
     5041 caggatcaat atataggcaa tatccaatat ggccctatgc caatatggct attggccagg
     5101 ttcaatacta tgtattggcc ctatgccata tagtattcca tatatgggtt ttcctattga
     5161 cgtagatagc ccctcccaat gggcggtccc atataccata tatggggctt cctaataccg
     5221 cccatagcca ctcccccatt gacgtcaatg gtctctatat atggtctttc ctattgacgt
     5281 catatgggcg gtcctattga cgtatatggc gcctccccca ttgacgtcaa ttacggtaaa
     5341 tggcccgcct ggctcaatgc ccattgacgt caataggacc acccaccatt gacgtcaatg
     5401 ggatggctca ttgcccattc atatccgttc tcacgccccc tattgacgtc aatgacggta
     5461 aatggcccac ttggcagtac atcaatatct attaatagta acttggcaag tacattacta
     5521 ttggaagtac gccagggtac attggcagta ctcccattga cgtcaatggc ggtaaatggc
     5581 ccgcgatggc tgccaagtac atccccattg acgtcaatgg ggaggggcaa tgacgcaaat
     5641 gggcgttcca ttgacgtaaa tgggcggtag gcgtgcctaa tgggaggtct atataagcaa
     5701 tgctcgttta gggaac
//
