LOCUS       sequence-268615-        4784 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Lifeact-GFP-ePDZ mRNA for microinjection to be used 
            for optogenetics in S. purpuratus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4784)
  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 4784)
  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-ePDZ
FEATURES             Location/Qualifiers
     source          1..4784
                     /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             883..933
                     /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"
     CDS             join(961..963,964..966,967..1677)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(961..963,964..966,967..1677)
                     /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(1006..1027)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1267..1286)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1614..1635
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2355..2375
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2388..2432
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     3'UTR           2477..2602
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(2658..2674)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2658..2674)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2671..2693)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2682..2698
                     /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(2706..2712,2713..2730,2731..2736))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2751..2772
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2889..2906)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3060..3648)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3140..3159)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3819..4610,4611..4679))
                     /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     4442..4461
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4680..4784)
                     /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 cagatctatg ccatcggggc ccatgggtgt cgcagatttg
      901 atcaagaaat tcgaaagcat ctcaaaggaa gaaggggatc caccggtcgc cagggccccc
      961 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1021 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1081 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1141 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1201 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1261 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1321 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1381 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1441 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1501 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1561 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1621 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct atacaagtcc
     1681 ggaggggatc ggtggagctc caccggtggc ggccgcatgc cagaacttgg atttagcata
     1741 tcaggtggtg tcgggggtag aggaaaccca ttcagacctg atgatgatgg tatatttgta
     1801 acaagggtac aacctgaagg accagcatca aaattactgc agccaggtga taaaattatt
     1861 caggctaatg gctacagttt tataaatatt gaacatggac aagcagtgtc cttgctaaaa
     1921 actttccaga atacagttga actcatcatt gtacgagaag ttggtaacgg tgcaaaacaa
     1981 gagattcgag tgagggttga aaaagacggt ggttctggag gcgtttcttc tgttccgacc
     2041 aatctggaag ttgttgctgc gaccccgact agcctgctga tcagctggga tgcgtatagg
     2101 gagcttccgg ttagttatta ccgtatcacg tacggtgaaa ccggtggtaa ctccccggtt
     2161 caggaattca ctgtacctgg ttccaagtct actgctacca tcagcggcct gaaaccgggt
     2221 gtcgactata ccatcactgt atacgcacat tacaactacc attactactc tagcccaatc
     2281 tcgattaact accgtacctc tagactcgag ctcaagcttc gaattctgca gtcgacggta
     2341 gcggccgctc tagagagaat ctttattttc agggcgccgc caaattcaaa gaaaccgctg
     2401 ctgctaaatt cgaacgccag cacatggaca gcggaggtgg aggtactagt gactgactag
     2461 gatctggtta ccactaaacc agcctcaaga acacccgaat ggagtctcta agctacataa
     2521 taccaactta cactttacaa aatgttgtcc cccaaaatgt agccattcgt atctgctcct
     2581 aataaaaaga aagtttcttc accccccctg caggtcgact ctagaggatc cccgggcgag
     2641 ctcgaattcg taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2701 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2761 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2821 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2881 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2941 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3001 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3061 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3121 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3181 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3241 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3301 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3361 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3421 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3481 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3541 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3601 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3661 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3721 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3781 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3841 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3901 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3961 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4021 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4081 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4141 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4201 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4261 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4321 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4381 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4441 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4501 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4561 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4621 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4681 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4741 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcg
//
