LOCUS       sequence-169691-        3826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway entry vector for expression of EGFP-tagged Lifeact
            in cells including neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3826)
  AUTHORS   Padilla-Rodriguez M, Parker SS, Adams DG, Westerling T, Puleo JI, 
            Watson AW, Hill SM, Noon M, Gaudin R, Aaron J, Tong D, Roe DJ, 
            Knudsen B, Mouneimne G
  TITLE     The actin cytoskeletal architecture of estrogen receptor positive 
            breast cancer cells suppresses invasion.
  JOURNAL   Nat Commun. 2018 Jul 30;9(1):2980. doi: 10.1038/s41467-018-05367-2.
  PUBMED    30061623
REFERENCE   2  (bases 1 to 3826)
  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: "2018-07-30"; volume: "9(1):2980. doi"; pages: " 
            10.1038/s41467-018-05367-2"
COMMENT     Sequence Label: pENTR CMVie-Lifeact-EGFP L1-R5
FEATURES             Location/Qualifiers
     source          1..3826
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        784..987
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     938..958
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     984..1008
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1064..1073
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1070..1120
                     /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      1136..1145
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1142..1144,1145..1147,1148..1861)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1142..1144,1145..1147,1148..1858)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1187..1208)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1448..1467)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1795..1816
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1889..2012
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2067..2086)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2068..2086)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2091..2107)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2091..2107)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2209..2228)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2220..3029
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2287..2306)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3176..3764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3665..3684
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctgg agacccaagc ttggtaccga gctcggatcc actagtatta tgcccagtac
      721 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      781 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      841 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      901 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      961 cggtgggagg tttatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc
     1021 catccacgct gttttgacct ccatagaaga ttctagagct agcgccacca tgggcgtggc
     1081 cgacttgatc aagaagttcg agtccatctc caaggaggag ggggatccac cggtcgccac
     1141 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1201 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1261 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1321 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1381 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1441 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1501 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1561 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1621 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1681 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1741 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1801 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     1861 aagcggcctc gagcatgcat ctagaggaca actttgtata caaaagttga acgagaaacg
     1921 taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa
     1981 tactgtaaaa cacaacatat ccagtcacta tgaatcaact acttagatgg tattagtgac
     2041 ctgtactgca gggcggccgc gatatcccct atagtgagtc gtattacatg gtcatagctg
     2101 tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa
     2161 atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     2221 tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt
     2281 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct
     2341 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     2401 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     2461 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     2521 gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     2581 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     2641 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     2701 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     2761 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     2821 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     2881 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     2941 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     3001 ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg taacactggc
     3061 agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga
     3121 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3181 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3241 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3301 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     3361 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3421 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3481 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3541 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3601 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3661 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3721 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3781 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt
//
