LOCUS       sequence-169690-        3819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway entry vector for expression of mRuby2-tagged 
            Lifeact in cells including neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3819)
  AUTHORS   Parker SS, Moutal A, Cai S, Chandrasekaran S, Roman MR, Koshy AA, 
            Khanna R, Zinsmaier KE, Mouneimne G
  TITLE     High Fidelity Cryopreservation and Recovery of Primary Rodent 
            Cortical Neurons.
  JOURNAL   eNeuro. 2018 Sep 27;5(5). pii: eN-MNT-0135-18. doi: 
            10.1523/ENEURO.0135-18.2018. eCollection 2018 Sep-Oct.
  PUBMED    30263951
REFERENCE   2  (bases 1 to 3819)
  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: "eNeuro."; 
            date: "2018-09-27"; volume: "5(5). pii: eN-MNT-0135-18. doi"; pages:
            " 10.1523/ENEURO.0135-18.2018. eCollection 2018 Sep-Oct"
COMMENT     Sequence Label: pENTR CMVie-Lifeact-mRuby2 L1-R5
FEATURES             Location/Qualifiers
     source          1..3819
                     /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..1852
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     protein_bind    1882..2005
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2060..2079)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2061..2079)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2084..2100)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2084..2100)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2202..2221)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2213..3022
                     /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(2280..2299)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3169..3757
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3658..3677
                     /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 catggtgtct aagggcgaag agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga
     1201 aggttcggtc aacggccacc aattcaaatg cacaggtgaa ggagaaggca atccgtacat
     1261 gggaactcaa accatgagga tcaaagtcat cgagggagga cccctgccat ttgcctttga
     1321 cattcttgcc acgtcgttca tgtatggcag ccgtactttt atcaagtacc cgaaaggcat
     1381 tcctgatttc tttaaacagt cctttcctga gggttttact tgggaaagag ttacgagata
     1441 cgaagatggt ggagtcgtca ccgtcatgca ggacaccagc cttgaggatg gctgtctcgt
     1501 ttaccacgtc caagtcagag gggtaaactt tccctccaat ggtcccgtga tgcagaagaa
     1561 gaccaagggt tgggagccta atacagagat gatgtatcca gcagatggtg gtctgagggg
     1621 atacactcat atggcactga aagttgatgg tggtggccat ctgtcttgct ctttcgtaac
     1681 aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg cccggtatcc atgccgttga
     1741 tcaccgcctg gaaaggttag aggaaagtga caatgaaatg ttcgtagtac aacgcgaaca
     1801 cgcagttgcc aagttcgccg ggcttggtgg tgggatggac gagctgtaca agtaagcggc
     1861 ctcgagcatg catctagagg acaactttgt atacaaaagt tgaacgagaa acgtaaaatg
     1921 atataaatat caatatatta aattagattt tgcataaaaa acagactaca taatactgta
     1981 aaacacaaca tatccagtca ctatgaatca actacttaga tggtattagt gacctgtact
     2041 gcagggcggc cgcgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg
     2101 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     2161 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     2221 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     2281 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     2341 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     2401 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     2461 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     2521 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     2581 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     2641 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     2701 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     2761 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     2821 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     2881 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     2941 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     3001 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     3061 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc
     3121 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3181 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3241 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3301 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3361 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3421 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3481 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3541 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3601 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3661 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3721 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3781 acggttcctg gccttttgct ggccttttgc tcacatgtt
//
