LOCUS       sequence-169689-        4031 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway entry vector for expression of iRFP670-tagged 
            Lifeact in cells including neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4031)
  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 4031)
  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-iRFP670 L1-R5
FEATURES             Location/Qualifiers
     source          1..4031
                     /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             1142..2077
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAG
                     AFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLH
                     RHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVM
                     LYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSR
                     IVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYE
                     PRAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     protein_bind    2094..2217
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2272..2291)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2273..2291)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2296..2312)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2296..2312)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2414..2433)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2425..3234
                     /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(2492..2511)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3381..3969
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3870..3889
                     /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 catggcgcgt aaggtcgatc tcacctcctg cgatcgcgag ccgatccaca tccccggcag
     1201 cattcagccg tgcggctgcc tgctagcctg cgacgcgcag gcggtgcgga tcacgcgcat
     1261 tacggaaaat gccggcgcgt tctttggacg cgaaactccg cgggtcggtg agctactcgc
     1321 cgattacttc ggcgagaccg aagcccatgc gctgcgcaac gcactggcgc agtcctccga
     1381 tccaaagcga ccggcgctga tcttcggttg gcgcgacggc ctgaccggcc gcaccttcga
     1441 catctcactg catcgccatg acggtacatc gatcatcgag ttcgagcctg cggcggccga
     1501 acaggccgac aatccgctgc ggctgacgcg gcagatcatc gcgcgcacca aagaactgaa
     1561 gtcgctcgaa gagatggccg cacgggtgcc gcgctatctg caggcgatgc tcggctatca
     1621 ccgcgtgatg ttgtaccgct tcgcggacga cggctccggg atggtgatcg gcgaggcgaa
     1681 gcgcagcgac ctcgagagct ttctcggtca gcactttccg gcgtcgctgg tcccgcagca
     1741 ggcgcggcta ctgtacttga agaacgcgat ccgcgtggtc tcggattcgc gcggcatcag
     1801 cagccggatc gtgcccgagc acgacgcctc cggcgccgcg ctcgatctgt cgttcgcgca
     1861 cctgcgcagc atctcgccct gccatctcga atttctgcgg aacatgggcg tcagcgcctc
     1921 gatgtcgctg tcgatcatca ttgacggcac gctatgggga ttgatcatct gtcatcatta
     1981 cgagccgcgt gccgtgccga tggcgcagcg cgtcgcggcc gaaatgttcg ccgacttctt
     2041 atcgctgcac ttcaccgccg cccaccacca acgctaagca tgcatctaga ggacaacttt
     2101 gtatacaaaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
     2161 tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt cactatgaat
     2221 caactactta gatggtatta gtgacctgta ctgcagggcg gccgcgatat cccctatagt
     2281 gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc
     2341 tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac
     2401 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga
     2461 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     2521 caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg
     2581 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     2641 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     2701 tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct
     2761 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     2821 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     2881 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     2941 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
     3001 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     3061 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     3121 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     3181 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa
     3241 ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc
     3301 tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta
     3361 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3421 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3481 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     3541 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3601 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3661 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3721 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3781 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3841 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3901 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3961 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4021 gctcacatgt t
//
