LOCUS       sequence-461771-        6330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP with a CAAX motif under control of a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6330)
  AUTHORS   Wilson MH, Hensley MR, Shen MC, Lu HY, Quinlivan VH, Busch-Nentwich 
            EM, Rawls JF, Farber SA
  TITLE     Zebrafish are resilient to the loss of major diacylglycerol 
            acyltransferase enzymes.
  JOURNAL   J Biol Chem. 2024 Nov 6;300(12):107973. doi: 
            10.1016/j.jbc.2024.107973.
  PUBMED    39510175
REFERENCE   2  (bases 1 to 6330)
  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"; journalName: "J Biol 
            Chem."; date: "2024-11-6"; volume: "300(12):107973. doi"; pages: " 
            10.1016/j.jbc.2024.107973"
COMMENT     Sequence Label: pDEST-CMV-EGFPCAAX
FEATURES             Location/Qualifiers
     source          1..6330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(17..36)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     364..385
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(507..531)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    533..667
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(730..750)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(758..774)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    790..924
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(1363..1550,1551..1562)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(1596..1612)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1596..1612)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1649..1669)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1649..1667)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1688..1704)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1701..1723)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1712..1728
                     /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(1736..1742,1743..1760,1761..1766))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1781..1802
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1919..1936)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2090..2678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2170..2189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2849..3640,3641..3709))
                     /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     3472..3491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3710..3814)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3977..3998)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(4347..4358,4359..4521)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     4894..4910
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4930..4950
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        4974..5958
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        5993..6011
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5993..6010
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    6011..6035
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(6041..6043,6044..6046,6047..6330,1..427)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6041..6043,6044..6046,6047..6330,1..427)
                     /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(6086..6107)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
       61 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
      121 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
      181 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
      241 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
      301 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
      361 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
      421 gtacaaggga ggaggaagat ctaagctgaa ccctcctgat gagagtggcc ccggctgcat
      481 gagctgcaag tgtgtgctct cctgagaccc agctttcttg tacaaagtgg gggatccaga
      541 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
      601 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
      661 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
      721 ggttttttcc aactttatta tacatagttg ataattcact ggccgtcgtt ttacggtacc
      781 atcgatgatg atccagacat gataagatac attgatgagt ttggacaaac cacaactaga
      841 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
      901 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
      961 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct
     1021 gattatgatc ctctagatca gatctgcgaa gatacggcca cgggtgctct tgatcctgtg
     1081 gctgattttg gactgtgctg ctcgcagctg ctgatgaatc acatacttcc tccattttct
     1141 tccactgatt gactgttata atttccctaa tttccaggtc aaggtgctgt gcattgtggt
     1201 aatagatgtg acatgacgtc acttccaaag gaccaatgaa catgtctgac caatttcata
     1261 taatgtgaaa acgattttca taggcagaat aaataacatt taaattaaac tgggcatcag
     1321 cgcaattcaa ttggtttggt aatagcaagg gaaaatagaa tgaagtgatc tccaaaaaat
     1381 aagtactttt tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa
     1441 aatgtaatta agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat
     1501 ccccaaaaat aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc
     1561 tggttcttga ccccctacct tcagcaagcc cagcagatcc actagttcta gagcggccgc
     1621 caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     1681 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     1741 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     1801 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     1861 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     1921 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1981 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2041 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2101 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2161 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2221 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2281 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2341 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2401 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2461 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2521 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2581 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     2641 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     2701 cggggtctga cgctcagtgg aacgaaaact cacgttaagg cattttggtc atgagattat
     2761 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     2821 gtatatatga gtaaacttgg tctgacagtt accaatgctt aataagtgag gcacctatct
     2881 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2941 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3001 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3061 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3121 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3181 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3241 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3301 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3361 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3421 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3481 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     3541 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     3601 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     3661 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     3721 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     3781 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     3841 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     3901 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     3961 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     4021 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca cgctggctaa gaactcatca
     4081 gcctccccgg tccatctacc cacgtaccaa tgcaccaatt ggccacaatg acggctacta
     4141 catggtgcca ttccttcctc tttataggaa tggagactac ctcctgtcca acaatgctct
     4201 tggatacgag tacgcctacc tgttggaccc aggtcattgc acaacaccag aaatgccctc
     4261 tgatctgcaa aagacgtgaa tatctgttca gacacccata tccactctgt tccacacagg
     4321 tcagaggttt gtccaggagt tcttgacaga ggtgtaaaaa gtactcaaaa attttactca
     4381 agtgaaagta caagtactta gggaaaattt tactcaatta aaagtaaaag tatctggcta
     4441 gaatcttact tgagtaaaag taaaaaagta ctccattaaa attgtacttg agtattaagg
     4501 aagtaaaagt aaaagcaaga aagaaaacta gagattcttg tttaagcttt taatctcaaa
     4561 aaacattaaa tgaaatgcat acaaggtttt atcctgcttt agaactgttt gtatttaatt
     4621 atcaaactat aagacagaca atctaatgcc agtacacgct actcaaagtt gtaaaacctc
     4681 agatttaact tcagtagaag ctgattctca aaattgttag tgtcaagcct agctcttttg
     4741 gggctgaaaa gcaatcctgc agtgctgaaa agcctctcac aggcagccga tgcgggaaga
     4801 ggtgtattag tcttgataga gaggctgcaa atagcaggaa acgtgagcag agactccctg
     4861 gtgtctgaaa cacaggccag atgggccctc gagcaggaaa cagctatgac catgattacg
     4921 ccaagctatc aactttgtat agaaaagttg aaggcctctt cgctattacg ccagtcgacc
     4981 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
     5041 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
     5101 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
     5161 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
     5221 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
     5281 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
     5341 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
     5401 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
     5461 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
     5521 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
     5581 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
     5641 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
     5701 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
     5761 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
     5821 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
     5881 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
     5941 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
     6001 gacactatag acaagtttgt acaaaaaagc aggctggacc atggtgagca agggcgagga
     6061 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     6121 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     6181 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     6241 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     6301 cgccatgccc gaaggctacg tccaggagcg
//
