LOCUS       sequence-268860-        4673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP-ePDZ-CAAX 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 4673)
  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 4673)
  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-GFP-ePDZ-CAAX
FEATURES             Location/Qualifiers
     source          1..4673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     5'UTR           1..43
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             join(66..68,69..71,72..782)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(66..68,69..71,72..782)
                     /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(111..132)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(372..391)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     719..740
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     3'UTR           1502..1627
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(1730..1746)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1730..1746)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1743..1765)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1754..1770
                     /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(1778..1784,1785..1802,1803..1808))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1823..1844
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1961..1978)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2132..2720)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2212..2231)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2891..3682,3683..3751))
                     /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     3514..3533
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3752..3856)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3924..3942
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3980..4002)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4102..4121
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4335..4354
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4644..4662
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4644..4661
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 cttgttcttt ttgcagaagc tcagaataaa cgctcaactt tggcagatct atgccatcgg
       61 ggcccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      121 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
      181 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
      241 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
      301 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
      361 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
      421 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
      481 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
      541 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
      601 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
      661 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
      721 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctataca
      781 agtccggagg ggatcggtgg agctccaccg gtggcggccg catgccagaa cttggattta
      841 gcatatcagg tggtgtcggg ggtagaggaa acccattcag acctgatgat gatggtatat
      901 ttgtaacaag ggtacaacct gaaggaccag catcaaaatt actgcagcca ggtgataaaa
      961 ttattcaggc taatggctac agttttataa atattgaaca tggacaagca gtgtccttgc
     1021 taaaaacttt ccagaataca gttgaactca tcattgtacg agaagttggt aacggtgcaa
     1081 aacaagagat tcgagtgagg gttgaaaaag acggtggttc tggaggcgtt tcttctgttc
     1141 cgaccaatct ggaagttgtt gctgcgaccc cgactagcct gctgatcagc tgggatgcgt
     1201 atagggagct tccggttagt tattaccgta tcacgtacgg tgaaaccggt ggtaactccc
     1261 cggttcagga attcactgta cctggttcca agtctactgc taccatcagc ggcctgaaac
     1321 cgggtgtcga ctataccatc actgtatacg cacattacaa ctaccattac tactctagcc
     1381 caatctcgat taactaccgt acctctagac tcgagaagga tggtaagaaa aagaagaaga
     1441 agtcaaaaac caagtgtgtt atcatgtaaa ctagtgactg actaggatct ggttaccact
     1501 aaaccagcct caagaacacc cgaatggagt ctctaagcta cataatacca acttacactt
     1561 tacaaaatgt tgtcccccaa aatgtagcca ttcgtatctg ctcctaataa aaagaaagtt
     1621 tcttcacatt ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaamcccccc cccccccccc
     1681 tgcaggtcga ctctagagga tccccgggcg agctcgaatt cgtaatcatg tcatagctgt
     1741 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     1801 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     1861 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     1921 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1981 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2041 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2101 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2161 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2221 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2281 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2341 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2401 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2461 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2521 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2581 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2641 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2701 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2761 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2821 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     2881 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2941 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3001 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3061 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3121 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3181 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3241 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3301 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3361 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3421 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3481 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3541 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3601 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3661 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3721 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3781 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3841 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     3901 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     3961 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     4021 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     4081 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccattcga
     4141 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     4201 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     4261 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     4321 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     4381 gtgatgccgg ccacgatgcg tccggcgtag aggatctggc tagcgatgac cctgctgatt
     4441 ggttcgctga ccatttccgg gtgcgggacg gcgttaccag aaactcagaa ggttcgtcca
     4501 accaaaccga ctctgacggc agtttacgag agagatgata gggtctgctt cagtaagcca
     4561 gatgctacac aattaggctt gtacatattg tcgttagaac gcggctacaa ttaatacata
     4621 accttatgta tcatacacat acgatttagg tgacactata gaatacaagc ttg
//
