LOCUS       sequence-169692-        3862 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Multisite Gateway entry vector for expression of mTagBFP2-tagged 
            Lifeact in cells including neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3862)
  TITLE     Lentiviral Lifeact
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3862)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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            "Unpublished"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-mTagBFP2 L1-R5
FEATURES             Location/Qualifiers
     source          1..3862
                     /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="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     protein_bind    1925..2048
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2103..2122)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2104..2122)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2127..2143)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2127..2143)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2245..2264)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2256..3065
                     /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(2323..2342)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3212..3800
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3701..3720
                     /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 agctgattaa ggagaacatg cacatgaagc tgtacatgga
     1201 gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga
     1261 gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga
     1321 catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat
     1381 ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata
     1441 cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
     1501 ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa
     1561 aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg
     1621 cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa acgccaagac
     1681 cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga
     1741 ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt
     1801 ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta attaagcggc
     1861 cgcgactcta gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag
     1921 aggacaactt tgtatacaaa agttgaacga gaaacgtaaa atgatataaa tatcaatata
     1981 ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag
     2041 tcactatgaa tcaactactt agatggtatt agtgacctgt actgcagggc ggccgcgata
     2101 tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt
     2161 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac
     2221 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     2281 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     2341 ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag
     2401 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     2461 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     2521 ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag
     2581 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     2641 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     2701 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     2761 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     2821 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     2881 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     2941 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     3001 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     3061 tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg
     3121 acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt
     3181 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3241 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3301 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     3361 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3421 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3481 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3541 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3601 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3661 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3721 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3781 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3841 gctggccttt tgctcacatg tt
//
