LOCUS       sequence-342356-        6795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for expression of MmCavin4-APEX2-P2A-mKate2 in mammalian 
            cells. Parton lab clone EJF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6795)
  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 6795)
  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: MmCavin4-APEX2-P2A-mKate2
FEATURES             Location/Qualifiers
     source          1..6795
                     /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"
     protein_bind    complement(1226..1250)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1255..2001
                     /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             2011..2067
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2086..2781
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     protein_bind    complement(2787..2807)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2847..2981
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2871..2890)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2925..2944
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3094..3114)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3094..3112)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3133..3149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3133..3149)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3146..3168)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3157..3173
                     /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(3181..3187,3188..3205,3206..3211))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3226..3247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3364..3381)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3535..4123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3615..3634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4294..5085,5086..5154))
                     /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     4917..4936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5155..5259)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5285..5740)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5422..5443)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5634..5653
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5811..6795
                     /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 ggaacacaac ggatcagctt caaatgctgg taaaatccac
      181 cagaatcggt tgtcaagtgt gacggaagat gaagaccaag acgcagccct caccattgtg
      241 accgtgctgg acagagtggc cagtgtcgtg gacagtgtgc aggcaagcca gaagagaatt
      301 gaggagagac acagagaaat gggtaacgcg ataaagtctg tccaaataga cctgctgaag
      361 ctctcacagt cacacagcaa tacgggctac gttgtcaaca agctgtttga gaagacccgg
      421 aaagtcagcg ctcacattaa ggatgtcaag gcccgggtag agaagcaaca ggttcgtgta
      481 accaaagtcg aaaccaagca agaagaaata atgaagaaaa acaaattccg agtggtaatc
      541 ttccaggagg acattccctg ccccgcatcc ctgtctgttg ttaaagacag aagcctgccc
      601 gagaaccagg aggaggccga agaagtcttc gatcccccaa tagagctctc ctcggatgag
      661 gagtattatg ttgaagaaag cagatctgcc aggcttagga agtcaggcaa agagcacatt
      721 gaccacatca agaaagcctt ttccagagaa aacatgcaga agacacggca gactcttgac
      781 aagaaagtga gtgggattag aactaggata gtcactcctg agaggagaga gaggctgagg
      841 cagtcgggag agaggctgag gcagtcagga gagaggctga ggcagtcggg ggaaagattt
      901 aagaaatcga tttccagtgc cgctccctca aaggaagctt ttaagattcg gagccttagg
      961 aaagcgaagg atcccaaggc cgagggccag gaggtagaca gagggatggg cgtggacatc
     1021 atctcaggta gcctggctct ggggcccatc catgagttcc actctgatga gttcagtgaa
     1081 acagaaaagg aggtgaccaa aggagggtac agtccccaag aaggagggga ccccccaacg
     1141 cctgagcctt tgaaggtgac ttttaaaccc caggtgaggg tagaggatga cgagtcactc
     1201 ctgttggagc taaagcagtc ctcataccca gctttcttgt acaaagtggt cgacggaaag
     1261 tcttacccaa ctgtgagtgc tgattaccag gacgccgttg agaaggcgaa gaagaagctc
     1321 agaggcttca tcgctgagaa gagatgcgct cctctaatgc tccgtttggc attccactct
     1381 gctggaacct ttgacaaggg cacgaagacc ggtggaccct tcggaaccat caagcaccct
     1441 gccgaactgg ctcacagcgc taacaacggt cttgacatcg ctgttaggct tttggagcca
     1501 ctcaaggcgg agttccctat tttgagctac gccgatttct accagttggc tggcgttgtt
     1561 gccgttgagg tcacgggtgg acctaaggtt ccattccacc ctggaagaga ggacaagcct
     1621 gagccaccac cagagggtcg cttgcccgat cccactaagg gttctgacca tttgagagat
     1681 gtgtttggca aagctatggg gcttactgac caagatatcg ttgctctatc tgggggtcac
     1741 actattggag ctgcacacaa ggagcgttct ggatttgagg gtccctggac ctctaatcct
     1801 cttattttcg acaactcata cttcacggag ttgttgagtg gtgagaagga aggtctcctt
     1861 cagctacctt ctgacaaggc tcttttgtct gaccctgtat tccgccctct cgttgacaaa
     1921 tatgcagcgg acgaagatgc cttctttgct gattacgctg aggctcacca aaagctttcc
     1981 gagcttgggt ttgctgatgc cggatccgga gccacgaact tctctctgtt aaagcaagca
     2041 ggagacgtgg aagaaaaccc cggtcctgct cctggaagcg gtaccgtgag cgagctgatt
     2101 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag
     2161 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggcg
     2221 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc
     2281 agcaaaacct tcatcaacca cacccagggc atccccgact tctttaagca gtccttcccc
     2341 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     2401 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     2461 ttcccatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc ctccaccgag
     2521 accctgtacc ccgctgacgg cggcctggaa ggcagagccg acatggccct gaagctcgtg
     2581 ggcgggggcc acctgatctg caacttgaag accacataca gatccaagaa acccgctaag
     2641 aacctcaaga tgcccggcgt ctactatgtg gacagaagac tggaaagaat caaggaggcc
     2701 gacaaagaga cctacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct
     2761 agcaaactgg ggcacagatg actagtcaac tttattatac atagttgcac tcgagcctct
     2821 agaactatag tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg
     2881 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     2941 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3001 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg gccgcggcgc
     3061 caatgcattg ggcccggtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
     3121 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3181 caacatacga gccgggagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3241 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3301 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3361 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3421 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3481 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3541 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3601 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3661 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3721 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3781 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3841 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3901 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3961 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4021 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4081 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4141 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4201 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4261 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4321 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4381 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4441 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4501 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4561 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4621 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4681 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4741 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4801 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4861 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4921 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4981 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5041 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5101 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5161 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5221 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5281 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5341 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5401 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5461 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5521 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5581 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5641 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5701 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     5761 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccgcc
     5821 aattcaatat ggcgtatatg gactcatgcc aattcaatat ggtggatctg gacctgtgcc
     5881 aattcaatat ggcgtatatg gactcgtgcc aattcaatat ggtggatctg gaccccagcc
     5941 aattcaatat ggcggacttg gcaccatgcc aattcaatat ggcggacctg gcactgtgcc
     6001 aactggggag gggtctactt ggcacggtgc caagtttgag gaggggtctt ggccctgtgc
     6061 caagtccgcc atattgaatt ggcatggtgc caataatggc ggccatattg gctatatgcc
     6121 aggatcaata tataggcaat atccaatatg gccctatgcc aatatggcta ttggccaggt
     6181 tcaatactat gtattggccc tatgccatat agtattccat atatgggttt tcctattgac
     6241 gtagatagcc cctcccaatg ggcggtccca tataccatat atggggcttc ctaataccgc
     6301 ccatagccac tcccccattg acgtcaatgg tctctatata tggtctttcc tattgacgtc
     6361 atatgggcgg tcctattgac gtatatggcg cctcccccat tgacgtcaat tacggtaaat
     6421 ggcccgcctg gctcaatgcc cattgacgtc aataggacca cccaccattg acgtcaatgg
     6481 gatggctcat tgcccattca tatccgttct cacgccccct attgacgtca atgacggtaa
     6541 atggcccact tggcagtaca tcaatatcta ttaatagtaa cttggcaagt acattactat
     6601 tggaagtacg ccagggtaca ttggcagtac tcccattgac gtcaatggcg gtaaatggcc
     6661 cgcgatggct gccaagtaca tccccattga cgtcaatggg gaggggcaat gacgcaaatg
     6721 ggcgttccat tgacgtaaat gggcggtagg cgtgcctaat gggaggtcta tataagcaat
     6781 gctcgtttag ggaac
//
