LOCUS       sequence-268617-        4039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Lifeact-GFP mRNA for microinjection for visualization 
            of actin in S. purpuratus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4039)
  AUTHORS   Uchida A, Yajima M
  TITLE     An optogenetic approach to control protein localization during 
            embryogenesis of the sea urchin.
  JOURNAL   Dev Biol. 2018 Sep 1;441(1):19-30. doi: 10.1016/j.ydbio.2018.06.015.
            Epub 2018 Jun 26.
  PUBMED    29958898
REFERENCE   2  (bases 1 to 4039)
  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"; doi: 
            "10.1016/j.ydbio.2018.06.015"; journalName: "Dev Biol"; date: 
            "2018-09-1- 1"; volume: "441"; issue: "1"; pages: "19-30"
COMMENT     Sequence Label: pSP64-Lifeact-GFP
FEATURES             Location/Qualifiers
     source          1..4039
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..86
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(124..146)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     246..265
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     479..498
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        788..806
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     788..805
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     5'UTR           818..860
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             886..936
                     /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      952..961
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(958..960,961..963,964..1674)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(958..960,961..963,964..1674)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1003..1024)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1264..1283)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1611..1632
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1681..1698
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1913..1929)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1913..1929)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1926..1948)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1937..1953
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1961..1967,1968..1985,1986..1991))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2006..2027
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2144..2161)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2315..2903)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2395..2414)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3074..3865,3866..3934))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3697..3716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3935..4039)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
       61 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
      121 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
      181 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
      241 ctatgcggca tcagagcaga ttgtactgag agtgcaccat tcgacgctct cccttatgcg
      301 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
      361 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
      421 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
      481 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
      541 tgcgtccggc gtagaggatc tggctagcga tgaccctgct gattggttcg ctgaccattt
      601 ccgggtgcgg gacggcgtta ccagaaactc agaaggttcg tccaaccaaa ccgactctga
      661 cggcagttta cgagagagat gatagggtct gcttcagtaa gccagatgct acacaattag
      721 gcttgtacat attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac
      781 acatacgatt taggtgacac tatagaatac aagcttgctt gttctttttg cagaagctca
      841 gaataaacgc tcaactttgg cagatctgat catgccatgg ggcccatggg tgtcgcagat
      901 ttgatcaaga aattcgaaag catctcaaag gaagaagggg atccaccggt cgccaccatg
      961 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1021 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1081 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1141 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1201 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1261 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1321 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1381 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1441 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1501 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1561 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1621 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagaagggt
     1681 catcatcacc atcaccattg aactagtgac tgactaggat ctggttacca ctaaaccagc
     1741 ctcaagaaca cccgaatgga gtctctaagc tacataatac caacttacac tttacaaaat
     1801 gttgtccccc aaaatgtagc cattcgtatc tgctcctaat aaaaagaaag tttcttcccc
     1861 ccctgcaggt cgactctaga ggatccccgg gcgagctcga attcgtaatc atgtcatagc
     1921 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     1981 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     2041 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2101 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2161 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2221 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2281 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2341 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2401 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2461 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2521 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2581 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2641 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2701 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     2761 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2821 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2881 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2941 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3001 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3061 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3121 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3181 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3241 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3301 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3361 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3421 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3481 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3541 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3601 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3661 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3721 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3781 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3841 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     3901 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     3961 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4021 aacaaatagg ggttccgcg
//
