LOCUS       sequence-269568-        5594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of EMTB-GFP-ePDZ mRNA for microinjection for use of 
            optogenetics in S. purpuratus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5594)
  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 5594)
  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-EMTB-GFP-ePDZ
FEATURES             Location/Qualifiers
     source          1..5594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     173..191
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(229..251)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     351..370
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     584..603
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        893..911
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     893..910
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     5'UTR           923..965
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             978..1007
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             join(1840..1842,1843..2553)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1840..1842,1843..2553)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1882..1903)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2143..2162)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2490..2511
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3231..3251
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3264..3308
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     3'UTR           3353..3478
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(3573..3589)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3573..3589)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3586..3608)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3597..3613
                     /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(3621..3627,3628..3645,3646..3651))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3666..3687
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3804..3821)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3975..4563)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4055..4074)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4734..5525,5526..5594))
                     /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     5357..5376
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
      181 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
      241 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      301 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      361 gcagattgta ctgagagtgc accattcgac gctctccctt atgcgactcc tgcattagga
      421 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
      481 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
      541 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
      601 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
      661 ggatctggct agcgatgacc ctgctgattg gttcgctgac catttccggg tgcgggacgg
      721 cgttaccaga aactcagaag gttcgtccaa ccaaaccgac tctgacggca gtttacgaga
      781 gagatgatag ggtctgcttc agtaagccag atgctacaca attaggcttg tacatattgt
      841 cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cgatttaggt
      901 gacactatag aatacaagct tgcttgttct ttttgcagaa gctcagaata aacgctcaac
      961 tttggcagat ctgaatggag cagaagctca tctcagaaga agacctcggc gcgccagtgc
     1021 gaagcgaaac agcacccgac agctacaaag tgcaagataa gaaaaatgcc tccagccgcc
     1081 ctgcctctgc aatttcagga caaaataaca accactcagg aaataaacca gaccctccgc
     1141 ctgtgttacg tgttgatgac cggcagcggc tggcccggga gcgacgtgag gaacgggaga
     1201 aacagctagc tgcaagagaa atagtgtggt tagaaagaga agagcgagcc aggcagcact
     1261 acgagaagca cctggaagag cggaagaaga ggttggagga gcagaggcag aaggaggagc
     1321 ggaggagggc tgctgtggag gagaagcgga ggcagagact tgaggaggac aaagaacgcc
     1381 acgaagctgt tgtacggcgc acaatggaaa ggagccagaa gccaaaacag aagcataacc
     1441 gttggtcgtg gggaggctct ctccatggga gccctagcat ccacagtgca gatccagaca
     1501 ggcggtcagt ttccaccatg aatctttcga aatatgttga tcccgtcatt agcaagcggc
     1561 tctcctcttc atctgcaact ttactaaatt ctccagatag agctcgccgc ctgcagctca
     1621 gcccatggga gagcagcgtt gttaacagac tcctgacgcc cacacattcg ttcctggcca
     1681 gaagtaaaag cacagctgcc ttgtctggag aagcagcatc ttgcagcccc atcatcatgc
     1741 cctacaaagc tgcacactct agaaattcga tggatcgacc aaaactcttt gtaacaccac
     1801 ctgagggctc ttcggaccca ccggtcgcca ccgggcccgg tgagcaaggg cgaggagctg
     1861 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1921 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     1981 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     2041 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2101 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2161 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2221 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     2281 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     2341 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     2401 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     2461 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2521 gggatcactc tcggcatgga cgagctatac aagtccggag gggatcggtg gagctccacc
     2581 ggtggcggcc gcatgccaga acttggattt agcatatcag gtggtgtcgg gggtagagga
     2641 aacccattca gacctgatga tgatggtata tttgtaacaa gggtacaacc tgaaggacca
     2701 gcatcaaaat tactgcagcc aggtgataaa attattcagg ctaatggcta cagttttata
     2761 aatattgaac atggacaagc agtgtccttg ctaaaaactt tccagaatac agttgaactc
     2821 atcattgtac gagaagttgg taacggtgca aaacaagaga ttcgagtgag ggttgaaaaa
     2881 gacggtggtt ctggaggcgt ttcttctgtt ccgaccaatc tggaagttgt tgctgcgacc
     2941 ccgactagcc tgctgatcag ctgggatgcg tatagggagc ttccggttag ttattaccgt
     3001 atcacgtacg gtgaaaccgg tggtaactcc ccggttcagg aattcactgt acctggttcc
     3061 aagtctactg ctaccatcag cggcctgaaa ccgggtgtcg actataccat cactgtatac
     3121 gcacattaca actaccatta ctactctagc ccaatctcga ttaactaccg tacctctaga
     3181 ctcgagctca agcttcgaat tctgcagtcg acggtagcgg ccgctctaga gagaatcttt
     3241 attttcaggg cgccgccaaa ttcaaagaaa ccgctgctgc taaattcgaa cgccagcaca
     3301 tggacagcgg aggtggaggt actagtgact gactaggatc tggttaccac taaaccagcc
     3361 tcaagaacac ccgaatggag tctctaagct acataatacc aacttacact ttacaaaatg
     3421 ttgtccccca aaatgtagcc attcgtatct gctccwaawa aaaaraaart ttcttcacaa
     3481 aaaaaaaaaa aaaaaaaaaa aaaaaamccc cccccccccc ccctgcaggt cgactctaga
     3541 ggatccccgg gcgagctcga attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt
     3601 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3661 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3721 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3781 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3841 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3901 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3961 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4021 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4081 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4141 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4201 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4261 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4321 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4381 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4441 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4501 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4561 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4621 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4681 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4741 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4801 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4861 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4921 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4981 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5041 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5101 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5161 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5221 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5281 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5341 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5401 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5461 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5521 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5581 tgttgaatac tcat
//
