LOCUS       sequence-70563-e        3359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contributes the coding sequence for EGFP as the 3'-module during 
            MultiSite Gateway-cloning of chimeric cDNAs encoding three-part 
            fusion proteins..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3359)
  AUTHORS   Buj R, Iglesias N, Planas AM, Santalucia T
  TITLE     A plasmid toolkit for cloning chimeric cDNAs encoding customized 
            fusion proteins into any Gateway destination expression vector.
  JOURNAL   BMC Mol Biol. 2013 Aug 20;14(1):18. doi: 10.1186/1471-2199-14-18.
  PUBMED    23957834
REFERENCE   2  (bases 1 to 3359)
  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: "BMC Mol 
            Biol."; date: "2013-08-20"; volume: "14(1):18. doi"; pages: " 
            10.1186/1471-2199-14-18"
COMMENT     Sequence Label: pDONR P2R-P3-EGFP
FEATURES             Location/Qualifiers
     source          1..3359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(750..752,753..755,756..1466)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKVSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(795..816)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1056..1075)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1403..1424
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1468..1563)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1600..1619)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1601..1619)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1624..1640)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1624..1640)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1742..1761)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1753..2562
                     /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(1820..1839)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2709..3297
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3198..3217
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtggaca tggtgagcaa gggcgaggag ctgttcaccg
      781 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag gtcagcgtgt
      841 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      901 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
      961 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1021 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1081 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1141 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1201 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     1261 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1321 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1381 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1441 ctctcggcat ggacgagctg tactaaacaa ctttattata caaagttggc attataaaaa
     1501 agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta
     1561 tttggagctc catggtagcg ttaacgcggc cgcgatatcc cctatagtga gtcgtattac
     1621 atggtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg atgttacatt
     1681 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat
     1741 acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac
     1801 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     1861 acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     1921 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     1981 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     2041 actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     2101 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     2161 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     2221 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     2281 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     2341 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     2401 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     2461 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     2521 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg
     2581 ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa
     2641 tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2701 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2761 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2821 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2881 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2941 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3001 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3061 cgaacgacct acaccgaact gagataccta cagcgtgagc attgagaaag cgccacgctt
     3121 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3181 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3241 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3301 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtt
//
