LOCUS       sequence-341961-        5647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lifeact-mEos2-APEX2 fusion for PALM and CLEM. Parton lab clone IET.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5647)
  AUTHORS   Hall TE, Martel N, Ariotti N, Xiong Z, Lo HP, Ferguson C, Rae J, Lim
            YW, Parton RG
  TITLE     In vivo cell biological screening identifies an endocytic capture 
            mechanism for T-tubule formation.
  JOURNAL   Nat Commun. 2020 Jul 24;11(1):3711. doi: 10.1038/s41467-020-17486-w.
  PUBMED    32709891
REFERENCE   2  (bases 1 to 5647)
  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: "Nat 
            Commun."; date: "2020-07-24"; volume: "11(1):3711. doi"; pages: " 
            10.1038/s41467-020-17486-w"
COMMENT     Sequence Label: Lifeact-mEos2-APEX2
FEATURES             Location/Qualifiers
     source          1..5647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..984
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1019..1037
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1019..1036
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    1092..1116
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1117..1126
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1123..1173
                     /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"
     protein_bind    complement(1850..1874)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2623..2643)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2683..2817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2707..2726)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2761..2780
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2930..2950)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2930..2948)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2969..2985)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2969..2985)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2982..3004)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2993..3009
                     /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(3017..3023,3024..3041,3042..3047))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3062..3083
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3200..3217)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3371..3959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3451..3470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4130..4921,4922..4990))
                     /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     4753..4772
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4991..5095)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5121..5576)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5258..5279)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5470..5489
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcgaccgcca attcaatatg gcgtatatgg actcatgcca attcaatatg gtggatctgg
       61 acctgtgcca attcaatatg gcgtatatgg actcgtgcca attcaatatg gtggatctgg
      121 accccagcca attcaatatg gcggacttgg caccatgcca attcaatatg gcggacctgg
      181 cactgtgcca actggggagg ggtctacttg gcacggtgcc aagtttgagg aggggtcttg
      241 gccctgtgcc aagtccgcca tattgaattg gcatggtgcc aataatggcg gccatattgg
      301 ctatatgcca ggatcaatat ataggcaata tccaatatgg ccctatgcca atatggctat
      361 tggccaggtt caatactatg tattggccct atgccatata gtattccata tatgggtttt
      421 cctattgacg tagatagccc ctcccaatgg gcggtcccat ataccatata tggggcttcc
      481 taataccgcc catagccact cccccattga cgtcaatggt ctctatatat ggtctttcct
      541 attgacgtca tatgggcggt cctattgacg tatatggcgc ctcccccatt gacgtcaatt
      601 acggtaaatg gcccgcctgg ctcaatgccc attgacgtca ataggaccac ccaccattga
      661 cgtcaatggg atggctcatt gcccattcat atccgttctc acgcccccta ttgacgtcaa
      721 tgacggtaaa tggcccactt ggcagtacat caatatctat taatagtaac ttggcaagta
      781 cattactatt ggaagtacgc cagggtacat tggcagtact cccattgacg tcaatggcgg
      841 taaatggccc gcgatggctg ccaagtacat ccccattgac gtcaatgggg aggggcaatg
      901 acgcaaatgg gcgttccatt gacgtaaatg ggcggtaggc gtgcctaatg ggaggtctat
      961 ataagcaatg ctcgtttagg gaaccgccat tctgcctggg gacgtcggag caagcttgat
     1021 ttaggtgaca ctatagaata caagctactt gttctttttg caggatccca tcgattcgaa
     1081 ttcaaggatc aacaagtttg tacaaaaaag caggctgcca ccatgggcgt ggccgacctg
     1141 atcaagaagt tcgagagcat cagcaaggag gagagtgcta tcaagccgga catgaaaatc
     1201 aagctgcgta tggaaggaaa tgtaaatgga catcactttg tcattgacgg tgatggcaca
     1261 gggaagccat tcgaagggaa gcagagtatg gacttggaag tcaaagaagg cggcccattg
     1321 cccttcgcgt ttgatattct gacgacggcg ttccattacg ggaaccgggt attcgcaaaa
     1381 tatcccgaca atatccaaga ttacttcaaa caatctttcc ccaaaggcta cagttgggag
     1441 cgttcactca cattcgagga tgggggaatc tgcatcgcgc gcaacgatat taccatggaa
     1501 ggtgacacct tctataataa agttaggttt tatggaacga acttccctgc taacggcccg
     1561 gtgatgcaaa agaaaacgct caaatgggaa cctagcaccg aaaagatgta cgtgagagac
     1621 ggtgtgctta ctggtgatat ccatatggct cttctcttgg aagggaatgc tcactataga
     1681 tgtgactttc gcactaccta taaggcgaaa gaaaagggag tgaagttgcc tggctatcat
     1741 tttgtagatc actgtataga gatattgtcc cacgataagg actataataa ggtgaaactg
     1801 tacgaacatg ccgtggcaca ctcaggcttg cccgataacg cgaggcgtca cccagctttc
     1861 ttgtacaaag tgggcaagag ctaccccacc gtgagcgccg actaccagga cgccgtggag
     1921 aaggccaaga agaagctgcg cggcttcatc gccgagaagc gctgcgcccc cctgatgctg
     1981 cgcctggcct tccacagcgc cggcaccttc gacaagggca ccaagaccgg cggccccttc
     2041 ggcaccatca agcaccccgc cgagctggcc cacagcgcca acaacggcct ggacatcgcc
     2101 gtgcgcctgc tggagcccct gaaggccgag ttccccatcc tgagctacgc cgacttctac
     2161 cagctggccg gcgtggtggc cgtggaggtg accggcggcc ccaaggtgcc cttccacccc
     2221 ggccgcgagg acaagcccga gccccccccc gagggccgcc tgcccgaccc caccaagggc
     2281 agcgaccacc tgcgcgacgt gttcggcaag gctatgggcc tgaccgacca ggacatcgtg
     2341 gccctgagcg gcggccacac catcggcgcc gcccacaagg agcgcagcgg cttcgagggc
     2401 ccctggacca gcaaccccct gatcttcgac aacagctact tcaccgagct gctgagcggc
     2461 gagaaggagg gcctgctgca gctgcccagc gacaaggccc tgctgagcga ccccgtgttc
     2521 cgccccctgg tggacaagta cgccgccgac gaggacgcct tcttcgccga ctacgccgag
     2581 gcccaccaga agctgagcga gctgggcttc gccgacgcct aacaacttta ttatacatag
     2641 ttgcactcga gcctctagaa ctatagtgag tcgtattacg tagatccaga catgataaga
     2701 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     2761 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     2821 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     2881 ttcgcggccg cggcgccaat gcattgggcc cggtacccag cttttgttcc ctttagtgag
     2941 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3001 cgctcacaat tccacacaac atacgagccg ggagcataaa gtgtaaagcc tggggtgcct
     3061 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3121 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3181 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3241 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3301 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3361 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3421 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3481 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3541 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3601 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3661 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3721 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3781 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3841 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3901 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3961 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4021 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4081 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4141 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4201 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4261 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4321 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4381 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4441 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4501 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4561 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4621 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4681 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4741 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4801 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4861 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4921 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4981 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5041 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5101 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5161 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5221 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5281 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5341 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5401 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5461 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     5521 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     5581 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     5641 acgccag
//
