LOCUS       sequence-385063-        3733 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway p3E 2A fluorophore.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3733)
  AUTHORS   Kemmler CL, Moran HR, Murray BF, Scoresby A, Klem JR, Eckert RL, 
            Lepovsky E, Bertho S, Nieuwenhuize S, Burger S, D'Agati G, Betz C, 
            Puller AC, Felker A, Ditrychova K, Botschi S, Affolter M, Rohner N, 
            Lovely CB, Kwan KM, Burger A, Mosimann C
  TITLE     Next-generation plasmids for transgenesis in zebrafish and beyond.
  JOURNAL   Development. 2023 Mar 28:dev.201531. doi: 10.1242/dev.201531.
  PUBMED    36975217
REFERENCE   2  (bases 1 to 3733)
  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: 
            "Development. 2023 Mar 28:dev.201531. doi: 10.1242/dev.201531."
COMMENT     Sequence Label: 459 p3E-2A-nlsEGFPpA
FEATURES             Location/Qualifiers
     source          1..3733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(88..211)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             223..279
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             286..306
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(352..354,355..357,358..1071)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(352..354,355..357,358..1068)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(397..418)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(658..677)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1005..1026
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1109..1243
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1133..1152)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1187..1206
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(1306..1401)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1439..1457)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1462..1478)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1462..1478)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1580..1599)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1591..2400
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1658..1677)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2547..3135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3036..3055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3289..3306
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3465..3492)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3584..3670)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(3719..3733,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(3733,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cctgcagctc tagagctcga attctacagg
       61 tcactaatac catctaagta gttggttcat agtgactgca tatgttgtgt tttacagtat
      121 tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac
      181 gtttctcgtt caactttctt gtacaaagtg gggggatccg gagccacgaa cttctctctg
      241 ttaaagcaag caggagacgt ggaagaaaac cccggtccta tggctccaaa gaagaagcgt
      301 aaggtaaatc gatcgaattc aaggcctctc gagcctctag aaggtggcgg aatggtgagc
      361 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
      421 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
      481 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
      541 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
      601 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
      661 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
      721 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
      781 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
      841 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
      901 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
      961 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1021 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aggcgcgcct
     1081 ctagaactat agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt
     1141 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     1201 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     1261 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttccaact ttattataca
     1321 aagttggcat tataaaaaag cattgcttat caatttgttg caacgaacag gtcactatca
     1381 gtcaaaataa aatcattatt tggagctcca tggtagcgtt aacgcggccg cgatatcccc
     1441 tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa
     1501 aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct
     1561 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg
     1621 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     1681 gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg
     1741 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     1801 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     1861 gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa
     1921 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     1981 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     2041 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     2101 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     2161 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     2221 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     2281 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     2341 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     2401 tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc
     2461 gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac
     2521 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2581 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2641 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     2701 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2761 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2821 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2881 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2941 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3001 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3061 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3121 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3181 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3241 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3301 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     3361 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     3421 attaatacgc gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca
     3481 ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc
     3541 ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga
     3601 gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc
     3661 gttttatttg atgcctggca gttccctact ctcgcgttaa cgctagcatg gatgttttcc
     3721 cagtcacgac gtt
//
