LOCUS       sequence-331462-        4872 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express ER-EmEGFP in zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4872)
  AUTHORS   Morishita H, Eguchi T, Tsukamoto S, Sakamaki Y, Takahashi S, Saito 
            C, Koyama-Honda I, Mizushima N
  TITLE     Organelle degradation in the lens by PLAAT phospholipases.
  JOURNAL   Nature. 2021 Apr;592(7855):634-638. doi: 10.1038/s41586-021-03439-w.
            Epub 2021 Apr 14.
  PUBMED    33854238
REFERENCE   2  (bases 1 to 4872)
  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: 
            "10.1038/s41586-021-03439-w"; journalName: "Nature"; date: 
            "2021-04"; volume: "592"; issue: "7855"; pages: "634-638"
COMMENT     Sequence Label: pCS2-ER-EmEGFP
FEATURES             Location/Qualifiers
     source          1..4872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1013..1031
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1013..1030
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             join(1123..1125,1126..1836)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKV
                     NFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1165..1186)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1426..1445)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1773..1794
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1902..2036
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1926..1945)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1980..1999
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2149..2169)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2149..2167)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2188..2204)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2188..2204)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2201..2223)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2212..2228
                     /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(2236..2242,2243..2260,2261..2266))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2281..2302
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2419..2436)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2590..3178)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2670..2689)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3349..4140,4141..4209))
                     /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     3972..3991
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4210..4314)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4340..4795)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4477..4498)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4689..4708
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
     1021 gacactatag aatacaagct acttgttctt tttgcaggat ccaccatgct gctatccgtg
     1081 cctctgctcc tgggcctgct gggcctggcc gctgccggag gcgtgagcaa gggcgaggag
     1141 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1201 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1261 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcacctac
     1321 ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1381 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1441 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1501 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1561 agccacaagg tctatatcac cgccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1621 acccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1681 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     1741 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1801 gccgggatca ctctcggcat ggacgagctg tacaagaagg acgagctgta agaattcaag
     1861 gcctctcgag cctctagaac tatagtgagt cgtattacgt agatccagac atgataagat
     1921 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     1981 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     2041 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat
     2101 tcgcggccgc ggcgccaatg cattgggccc ggtacccagc ttttgttccc tttagtgagg
     2161 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2221 gctcacaatt ccacacaaca tacgagccgg gagcataaag tgtaaagcct ggggtgccta
     2281 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2341 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2401 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     2461 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2521 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2581 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2641 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2701 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2761 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2821 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2881 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2941 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3001 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3061 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3121 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3181 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3241 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3301 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3361 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3421 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     3481 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     3541 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     3601 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     3661 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     3721 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     3781 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     3841 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     3901 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     3961 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4021 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4081 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4141 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4201 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4261 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4321 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     4381 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     4441 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     4501 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     4561 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     4621 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     4681 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     4741 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     4801 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     4861 cgccagtcga cc
//
