LOCUS       sequence-457422-        5987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of LifeAct tagged with StayGold in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5987)
  AUTHORS   Winkelmann H, Richter CP, Eising J, Piehler J, Kurre R
  TITLE     Correlative single-molecule and structured illumination microscopy 
            of fast dynamics at the plasma membrane.
  JOURNAL   Nat Commun. 2024 Jul 10;15(1):5813. doi: 10.1038/s41467-024-49876-9.
  PUBMED    38987559
REFERENCE   2  (bases 1 to 5987)
  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: "2024-07-10"; volume: "15(1):5813. doi"; pages: " 
            10.1038/s41467-024-49876-9"
COMMENT     Sequence Label: pSems LifeAct-StayGold
FEATURES             Location/Qualifiers
     source          1..5987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             918..968
                     /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"
     intron          1763..1992
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1975..1994
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2065..2639,2640..2642,2643..2651)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2232..2249)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2459..2478
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2674..3477
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2737..2756)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3347..3366
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3607..3728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3644..3663)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3830..3846)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3830..3846)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3843..3865)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3854..3870
                     /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(3878..3884,3885..3902,3903..3908))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3923..3944
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4061..4078)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4232..4820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4312..4331)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4991..5782,5783..5851))
                     /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     5614..5633
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5852..5956)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgatatcatg ggtgtcgcag atttgatcaa gaaattcgaa agcatctcaa
      961 aggaagaact cgagggccat ggcacaggct caacaggctc aggctcatca ggatccatgg
     1021 ccagcaccgg ggaggagctg ttcaccgggg tggtgccctt caagttccag ctgaagggca
     1081 ccatcaacgg caagagcttc accgtggaag gcgagggcga gggcaatagc cacgagggca
     1141 gccacaaagg caagtacgtg tgcaccagcg gcaaactgcc aatgtcttgg gccgccctgg
     1201 gaactagctt cggctatggc atgaagtact acaccaagta ccccagcggc ctgaagaact
     1261 ggttccacga ggtgatgccc gagggcttca cctacgacag acacatccag tacaagggcg
     1321 acggcagcat ccacgccaag caccagcact tcatgaagaa cggcacctac cacaacatcg
     1381 tggagttcac cggccaggac ttcaaggaga acagccccgt gctgaccggc gacatgaacg
     1441 tgagcctgcc caacgaggtg cagcacatcc ccagagatga cggcgtggag tgcccagtga
     1501 ccctgctgta ccctctgctg agcgacaaga gcaagtgcgt ggaggcccac cagaacacca
     1561 tctgcaagcc cctgcacaat cagccagccc ccgatgtgcc ataccactgg atcagaaagc
     1621 agtacaccca gagcaaggac gacaccgagg agagagacca catctgccag agcgagaccc
     1681 tggaggccca cctggcggcc gcatagataa ctgatccagt gtgctggaat taattcgctg
     1741 tctgcgaggg ccagctgttg gggtgagtac tccctctcaa aagcgggcat gacttctgcg
     1801 ctaagattgt cagtttccaa aaacgaggag gatttgatat tcacctggcc cgcggtgatg
     1861 cctttgaggg tggccgcgtc catctggtca gaaaagacaa tctttttgtt gtcaagcttg
     1921 aggtgtggca ggcttgagat ctggccatac acttgagtga caatgacatc cactttgcct
     1981 ttctctccac aggtgtccac tcccaggtcc aactgcaggt cgagcatgca tctagggcgg
     2041 ccaattccgc ccctctcccc cccccccctc tccctccccc ccccctaacg ttactggccg
     2101 aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc
     2161 gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag
     2221 gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt
     2281 tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa
     2341 ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc
     2401 aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg
     2461 gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat
     2521 gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa
     2581 acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataagc
     2641 ttgccacaac ccgggataat tcctgcagcc aatatgggat cggccattga acaagatgga
     2701 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     2761 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     2821 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     2881 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     2941 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3001 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3061 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3121 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3181 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgatga tctcgtcgtg
     3241 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     3301 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     3361 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     3421 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaggg
     3481 gatcaattct ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     3541 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     3601 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3661 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3721 gtatcttaac gcgtcgagtg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     3781 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     3841 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3901 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3961 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4021 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4081 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4141 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4201 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4261 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4321 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4381 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4441 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4501 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4561 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4621 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4681 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4741 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4801 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4861 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4921 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4981 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5041 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5101 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5161 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5221 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5281 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5341 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5401 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5461 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5521 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5581 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5641 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5701 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5761 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5821 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5881 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5941 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
