LOCUS       sequence-380068-        4785 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian transient expression - expression of Lifeact-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4785)
  AUTHORS   Herron JC, Hu S, Watanabe T, Nogueira AT, Liu B, Kern ME, Aaron J, 
            Taylor A, Pablo M, Chew TL, Elston TC, Hahn KM
  TITLE     Actin nano-architecture of phagocytic podosomes.
  JOURNAL   Nat Commun. 2022 Jul 27;13(1):4363. doi: 10.1038/s41467-022-32038-0.
  PUBMED    35896550
REFERENCE   2  (bases 1 to 4785)
  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: "2022-07-27"; volume: "13(1):4363. doi"; pages: " 
            10.1038/s41467-022-32038-0"
COMMENT     Sequence Label: Lifeact-mCherry
FEATURES             Location/Qualifiers
     source          1..4785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    609..665
                     /label=MCS
                     /note="multiple cloning site"
     CDS             669..719
                     /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"
     CDS             738..1448
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(883..901)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1151..1170
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1573..1694
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1610..1629)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1664..1683
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1701..2156)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1838..1859)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2050..2069
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2183..2287
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2289..2646
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2289..2480
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2312..2332)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2497..2632
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2681..3475
                     /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             2681..3475
                     /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"
     primer_bind     complement(2735..2754)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3345..3364
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3663..3682)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3707..3754
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4083..4671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4572..4591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccat gggtgttgca gacctaataa agaaattcga gtcaatctcg aaagaggagg
      721 gttccggacc atccggaatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg
      781 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
      841 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
      901 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
      961 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     1021 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     1081 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     1141 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
     1201 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
     1261 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
     1321 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca
     1381 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt
     1441 acaagtaata agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt
     1501 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     1561 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     1621 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     1681 atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     1741 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     1801 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     1861 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     1921 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     1981 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     2041 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     2101 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     2161 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2221 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2281 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     2341 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2401 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2461 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2521 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2581 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     2641 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     2701 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     2761 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     2821 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     2881 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     2941 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3001 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3061 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3121 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3181 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     3241 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3301 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3361 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3421 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     3481 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     3541 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3601 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     3661 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     3721 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     3781 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     3841 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     3901 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     3961 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4021 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4081 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4141 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4201 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4261 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4321 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4381 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4441 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4501 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4561 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4621 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4681 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4741 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
