LOCUS       sequence-169693-        3883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway entry vector for expression of mCardinal-tagged 
            Lifeact in cells including neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3883)
  TITLE     Lentiviral Lifeact
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3883)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pENTR CMVie-Lifeact-mCardinal L1-R5
FEATURES             Location/Qualifiers
     source          1..3883
                     /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..1876
                     /codon_start=1
                     /product="far-red fluorescent protein derived from 
                     mNeptune, with further red-shifted spectra"
                     /label=mCardinal
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTTEGEGKPYEGTQTQ
                     RIKVVEGGPLPFAFDILATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTVTQDTSLQDGCLIYNVKLRGVNFPSNGPVMQKKTLGWEATTETLYPADGGLEGRCD
                     MALKLVGGGHLHCNLKTTYRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     protein_bind    1946..2069
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2124..2143)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2125..2143)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2148..2164)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2148..2164)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2266..2285)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2277..3086
                     /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(2344..2363)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3233..3821
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3722..3741
                     /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 agctgatcaa ggagaacatg cacatgaagc tgtacatgga
     1201 aggcaccgtg aacaaccacc acttcaagtg caccaccgaa ggggagggca agccctacga
     1261 gggcacccag acccagagga ttaaggtggt ggagggaggc cccctgccgt tcgcattcga
     1321 catcctggcc acctgcttta tgtacgggag caagaccttc atcaaccaca cccagggcat
     1381 ccccgatttc tttaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata
     1441 cgaagacggg ggcgtgctta ccgttaccca ggacaccagc ctccaggacg gctgcttgat
     1501 ctacaacgtc aagctcagag gggtgaactt cccatccaac ggccctgtga tgcagaagaa
     1561 aacactcggc tgggaggcca ccaccgagac cctgtacccc gctgacggcg gcctggaagg
     1621 cagatgcgac atggccctga agctcgtggg cgggggccac ctgcactgca acctgaagac
     1681 cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct actttgtgga
     1741 ccgcagactg gaaagaatca aggaggccga caatgagacc tacgtcgagc agcacgaggt
     1801 ggctgtggcc agatactgcg acctccctag caaactgggg cacaaactta atggcatgga
     1861 cgagctgtac aagtaagcgg ccgcgactct agaattctgc agatatccat cacactggcg
     1921 gccgctcgag catgcatcta gaggacaact ttgtatacaa aagttgaacg agaaacgtaa
     1981 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
     2041 tgtaaaacac aacatatcca gtcactatga atcaactact tagatggtat tagtgacctg
     2101 tactgcaggg cggccgcgat atcccctata gtgagtcgta ttacatggtc atagctgttt
     2161 cctggcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
     2221 tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     2281 gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat
     2341 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt
     2401 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     2461 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     2521 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggaa
     2581 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     2641 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     2701 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     2761 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     2821 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     2881 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     2941 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     3001 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     3061 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga
     3121 gcattacgct gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt
     3181 acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3241 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3301 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3361 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3421 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3481 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3541 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3601 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3661 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3721 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3781 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3841 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtt
//
