LOCUS       sequence-375339-        4747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construct for labelling actin filaments in mammalian cells with 
            lifeact-mScarlet3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4747)
  AUTHORS   Gadella TWJ Jr, van Weeren L, Stouthamer J, Hink MA, Wolters AHG, 
            Giepmans BNG, Aumonier S, Dupuy J, Royant A
  TITLE     mScarlet3: a brilliant and fast-maturing red fluorescent protein.
  JOURNAL   Nat Methods. 2023 Apr;20(4):541-545. doi: 
            10.1038/s41592-023-01809-y. Epub 2023 Mar 27.
  PUBMED    36973546
REFERENCE   2  (bases 1 to 4747)
  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: 
            "10.1038/s41592-023-01809-y"; journalName: "Nat Methods"; date: 
            "2023-04"; volume: "20"; issue: "4"; pages: "541-545"
COMMENT     Sequence Label: pLifeAct-mScarlet3_N1
FEATURES             Location/Qualifiers
     source          1..4747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..50)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     641..660
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(959..978)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1003..1050
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1379..1967
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1868..1887
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2142..2445
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2446..2649
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2600..2620
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2720..2770
                     /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      2786..2795
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2792..3478
                     /codon_start=1
                     /product="bright cysteine-free monomeric red fluorescent 
                     protein with fast and complete maturation (Gadella et al., 
                     2023)"
                     /label=mScarlet3
                     /translation="MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LRVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGA
                     VSVAQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKM
                     ALRLKDGGRYLADFKTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHS
                     TGGSGGS"
     primer_bind     3190..3209
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3616..3737
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3653..3672)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3707..3726
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3744..4199)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3881..3902)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4093..4112
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4226..4330
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4332..4689
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4332..4523
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4355..4375)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4540..4675
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(4724..4747,1..771)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             join(4724..4747,1..771)
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
ORIGIN
        1 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
       61 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
      121 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
      181 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
      241 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
      301 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
      361 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
      421 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
      481 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
      541 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
      601 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
      661 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
      721 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
      781 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
      841 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
      901 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
      961 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     1021 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     1081 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     1141 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     1201 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     1261 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     1321 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     1381 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     1441 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     1501 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     1561 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     1621 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     1681 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1741 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1801 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1861 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     1921 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     1981 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     2041 tatcccctga ttctgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca
     2101 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     2161 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     2221 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     2281 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     2341 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     2401 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     2461 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     2521 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     2581 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     2641 agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggactca gatctcgagc
     2701 tcaagcttcg aattccacca tgggcgtggc cgacctgatc aagaagttcg agagcatcag
     2761 caaggaggag ggggatccac cggtcgccac catggatagc accgaggcag tgatcaagga
     2821 gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac ggccacgagt tcgagatcga
     2881 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga gggtgaccaa
     2941 gggtggcccc ctgcccttct cctgggacat cctgtcccct cagttcatgt acggctccag
     3001 ggccttcacg aagcaccccg ccgacatccc cgactactgg aagcagtcct tccccgaggg
     3061 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gccgtgtccg tggcccagga
     3121 cacctccctg gaggacggca ccctgatcta caaggtgaag ctccgcggca ccaacttccc
     3181 tcctgacggc cccgtaatgc agaagaagac aatgggctgg gaagcatcca ccgagcggtt
     3241 gtaccccgag gacgtcgtgc tgaagggcga cattaagatg gccctgcgcc tgaaggacgg
     3301 cggccgctac ctggcggact tcaagaccac ctacagggcc aagaagcccg tgcagatgcc
     3361 cggcgccttc aacatcgacc gcaagttgga catcacatcc cacaacgagg actacaccgt
     3421 ggtggaacag tacgaacgct ccgtggcccg ccactccacc ggcggctccg gtggctccta
     3481 actgtacaag taaagcggcc gcgactctag atcataatca gccataccac atttgtagag
     3541 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     3601 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3661 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3721 ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     3781 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     3841 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     3901 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     3961 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     4021 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     4081 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     4141 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     4201 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4261 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4321 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     4381 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4441 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4501 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4561 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4621 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4681 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     4741 attgcac
//
